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Angiogenesis
Angiogenesis following vasculogenesis
Identifiers
MeSHD000096482
Anatomical terminology
3D medical animation still showing angiogenesis

Angiogenesis is the physiological process through which new blood vessels form from pre-existing vessels,[1][2][3] formed in the earlier stage of vasculogenesis. Angiogenesis continues the growth of the vasculature mainly by processes of sprouting and splitting, but processes such as coalescent angiogenesis,[4] vessel elongation and vessel cooption also play a role.[2] Vasculogenesis is the embryonic formation of endothelial cells from mesoderm cell precursors,[5] and from neovascularization, although discussions are not always precise (especially in older texts). The first vessels in the developing embryo form through vasculogenesis, after which angiogenesis is responsible for most, if not all, blood vessel growth during development and in disease.[6][7][8]

Angiogenesis is a normal and vital process in growth and development, as well as in wound healing and in the formation of granulation tissue. However, it is also a fundamental step in the transition of tumors from a benign to malignant state, leading to the use of angiogenesis inhibitors in the treatment of cancer.[9] The essential role of angiogenesis in tumor growth was first proposed in 1971 by Judah Folkman, who described tumors as "hot and bloody,"[10] illustrating that, at least for many tumor types, flush perfusion and even hyperemia are characteristic.

Types

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Sprouting angiogenesis

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Sprouting angiogenesis was the first identified form of angiogenesis and because of this, it is much more understood than intussusceptive angiogenesis. It occurs in several well-characterized stages. The initial signal comes from tissue areas that are devoid of vasculature. The hypoxia that is noted in these areas causes the tissues to demand the presence of nutrients and oxygen that will allow the tissue to carry out metabolic activities. Because of this, parenchymal cells will secrete vascular endothelial growth factor (VEGF-A) which is a proangiogenic growth factor.[11] These biological signals activate receptors on endothelial cells present in pre-existing blood vessels. Second, the activated endothelial cells, also known as tip cells,[12] begin to release enzymes called proteases that degrade the basement membrane to allow endothelial cells to escape from the original (parent) vessel walls. The endothelial cells then proliferate into the surrounding matrix and form solid sprouts connecting neighboring vessels. The cells that are proliferating are located behind the tip cells and are known as stalk cells.[12] The proliferation of these cells allows the capillary sprout to grow in length simultaneously.

As sprouts extend toward the source of the angiogenic stimulus, endothelial cells migrate in tandem, using adhesion molecules called integrins. These sprouts then form loops to become a full-fledged vessel lumen as cells migrate to the site of angiogenesis. Sprouting occurs at a rate of several millimeters per day, and enables new vessels to grow across gaps in the vasculature. It is markedly different from splitting angiogenesis because it forms entirely new vessels as opposed to splitting existing vessels.

Intussusceptive angiogenesis

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Main article: Intussusceptive angiogenesis

Intussusceptive angiogenesis, also known as splitting angiogenesis, is the formation of a new blood vessel by splitting an existing blood vessel into two.

Intussusception was first observed in neonatal rats. In this type of vessel formation, the capillary wall extends into the lumen to split a single vessel in two. There are four phases of intussusceptive angiogenesis. First, the two opposing capillary walls establish a zone of contact. Second, the endothelial cell junctions are reorganized and the vessel bilayer is perforated to allow growth factors and cells to penetrate into the lumen. Third, a core is formed between the 2 new vessels at the zone of contact that is filled with pericytes and myofibroblasts. These cells begin laying collagen fibers into the core to provide an extracellular matrix for growth of the vessel lumen. Finally, the core is fleshed out with no alterations to the basic structure. Intussusception is important because it is a reorganization of existing cells. It allows a vast increase in the number of capillaries without a corresponding increase in the number of endothelial cells. This is especially important in embryonic development as there are not enough resources to create a rich microvasculature with new cells every time a new vessel develops.[13]

Coalescent angiogenesis

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Main article: Coalescent angiogenesis

Coalescent angiogenesis is a mode of angiogenesis, considered to be the opposite of intussusceptive angiogenesis, where capillaries fuse, or coalesce, to make a larger bloodvessel, thereby increasing blood flow and circulation.[14] Coalescent angiogenesis has extended out of the domain of embryology. It is assumed to play a role in the formation of neovasculature, such as in a tumor.[15]

Physiology

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Mechanical stimulation

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Mechanical stimulation of angiogenesis is not well characterized. There is a significant amount of controversy with regard to shear stress acting on capillaries to cause angiogenesis, although current knowledge suggests that increased muscle contractions may increase angiogenesis.[16] This may be due to an increase in the production of nitric oxide during exercise. Nitric oxide results in vasodilation of blood vessels.

Chemical stimulation

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Chemical stimulation of angiogenesis is performed by various angiogenic proteins e.g. integrins and prostaglandins, including several growth factors e.g. VEGF, FGF.

Overview

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Stimulator Mechanism
FGF Promotes proliferation & differentiation of endothelial cells, smooth muscle cells, and fibroblasts
VEGF Affects permeability
VEGFR and NRP-1 Integrate survival signals
Ang1 and Ang2 Stabilize vessels
PDGF (BB-homodimer) and PDGFR recruit smooth muscle cells
TGF-β, endoglin and TGF-β receptors ↑extracellular matrix production
CCL2 Recruits lymphocytes to sites of inflammation
Histamine
Integrins αVβ3, αVβ5 (?[17]) and α5β1 Bind matrix macromolecules and proteinases
VE-cadherin and CD31 endothelial junctional molecules
ephrin Determine formation of arteries or veins
plasminogen activators remodels extracellular matrix, releases and activates growth factors
plasminogen activator inhibitor-1 stabilizes nearby vessels
eNOS and COX-2
AC133 regulates angioblast differentiation
ID1/ID3 Regulates endothelial transdifferentiation
Class 3 semaphorins Modulates endothelial cell adhesion, migration, proliferation and apoptosis. Alters vascular permeability[18]
Nogo-A Regulates endothelial cell migration and proliferation.[19] Alters vascular permeability.[20]

FGF

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Further information: Fibroblast growth factor

The fibroblast growth factor (FGF) family with its prototype members FGF-1 (acidic FGF) and FGF-2 (basic FGF) consists to date of at least 22 known members.[21] Most are single-chain peptides of 16-18 kDa and display high affinity to heparin and heparan sulfate. In general, FGFs stimulate a variety of cellular functions by binding to cell surface FGF-receptors in the presence of heparin proteoglycans. The FGF-receptor family is composed of seven members, and all the receptor proteins are single-chain receptor tyrosine kinases that become activated through autophosphorylation induced by a mechanism of FGF-mediated receptor dimerization. Receptor activation gives rise to a signal transduction cascade that leads to gene activation and diverse biological responses, including cell differentiation, proliferation, and matrix dissolution, thus initiating a process of mitogenic activity critical for the growth of endothelial cells, fibroblasts, and smooth muscle cells. FGF-1, unique among all 22 members of the FGF family, can bind to all seven FGF-receptor subtypes, making it the broadest-acting member of the FGF family, and a potent mitogen for the diverse cell types needed to mount an angiogenic response in damaged (hypoxic) tissues, where upregulation of FGF-receptors occurs.[22] FGF-1 stimulates the proliferation and differentiation of all cell types necessary for building an arterial vessel, including endothelial cells and smooth muscle cells; this fact distinguishes FGF-1 from other pro-angiogenic growth factors, such as vascular endothelial growth factor (VEGF), which primarily drives the formation of new capillaries.[23][24]

Besides FGF-1, one of the most important functions of fibroblast growth factor-2 (FGF-2 or bFGF) is the promotion of endothelial cell proliferation and the physical organization of endothelial cells into tube-like structures, thus promoting angiogenesis. FGF-2 is a more potent angiogenic factor than VEGF or PDGF (platelet-derived growth factor); however, it is less potent than FGF-1. As well as stimulating blood vessel growth, aFGF (FGF-1) and bFGF (FGF-2) are important players in wound healing. They stimulate the proliferation of fibroblasts and endothelial cells that give rise to angiogenesis and developing granulation tissue; both increase blood supply and fill up a wound space/cavity early in the wound-healing process.

VEGF

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Vascular endothelial growth factor (VEGF) has been demonstrated to be a major contributor to angiogenesis, increasing the number of capillaries in a given network. Initial in vitro studies demonstrated bovine capillary endothelial cells will proliferate and show signs of tube structures upon stimulation by VEGF and bFGF, although the results were more pronounced with VEGF.[25] Upregulation of VEGF is a major component of the physiological response to exercise and its role in angiogenesis is suspected to be a possible treatment in vascular injuries.[26][27][28][29] In vitro studies clearly demonstrate that VEGF is a potent stimulator of angiogenesis because, in the presence of this growth factor, plated endothelial cells will proliferate and migrate, eventually forming tube structures resembling capillaries.[16] VEGF causes a massive signaling cascade in endothelial cells. Binding to VEGF receptor-2 (VEGFR-2) starts a tyrosine kinase signaling cascade that stimulates the production of factors that variously stimulate vessel permeability (eNOS, producing NO), proliferation/survival (bFGF), migration (ICAMs/VCAMs/MMPs) and finally differentiation into mature blood vessels. Mechanically, VEGF is upregulated with muscle contractions as a result of increased blood flow to affected areas. The increased flow also causes a large increase in the mRNA production of VEGF receptors 1 and 2. The increase in receptor production means muscle contractions could cause upregulation of the signaling cascade relating to angiogenesis. As part of the angiogenic signaling cascade, NO is widely considered to be a major contributor to the angiogenic response because inhibition of NO significantly reduces the effects of angiogenic growth factors. However, inhibition of NO during exercise does not inhibit angiogenesis, indicating there are other factors involved in the angiogenic response.[16]

Angiopoietins

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The angiopoietins, Ang1 and Ang2, are required for the formation of mature blood vessels, as demonstrated by mouse knock out studies.[30] Ang1 and Ang2 are protein growth factors which act by binding their receptors, Tie-1 and Tie-2; while this is somewhat controversial, it seems that cell signals are transmitted mostly by Tie-2; though some papers show physiologic signaling via Tie-1 as well. These receptors are tyrosine kinases. Thus, they can initiate cell signaling when ligand binding causes a dimerization that initiates phosphorylation on key tyrosines.

MMP

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Another major contributor to angiogenesis is matrix metalloproteinase (MMP). MMPs help degrade the proteins that keep the vessel walls solid. This proteolysis allows the endothelial cells to escape into the interstitial matrix as seen in sprouting angiogenesis. Inhibition of MMPs prevents the formation of new capillaries.[31] These enzymes are highly regulated during the vessel formation process because destruction of the extracellular matrix would decrease the integrity of the microvasculature.[16]

Dll4

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Delta-like ligand 4 (Dll4) is a protein with a negative regulatory effect on angiogenesis.[32][33] Dll4 is a transmembrane ligand, for the notch family of receptors. There have been many studies conducted that have served to determine consequences of the Delta-like Ligand 4. One study in particular evaluated the effects of Dll4 on tumor vascularity and growth.[34] In order for a tumor to grow and develop, it must have the proper vasculature. The VEGF pathway is vital to the development of vasculature that in turn, helps the tumors to grow. The combined blockade of VEGF and Dll4 results in the inhibition of tumor progression and angiogenesis throughout the tumor. This is due to the hindrance of signaling in endothelial cell signaling which cuts off the proliferation and sprouting of these endothelial cells. With this inhibition, the cells do not uncontrollably grow, therefore, the cancer is stopped at this point. if the blockade, however, were to be lifted, the cells would begin their proliferation once again.[35]

Class 3 semaphorins

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Class 3 semaphorins (SEMA3s) regulate angiogenesis by modulating endothelial cell adhesion, migration, proliferation, survival and the recruitment of pericytes.[18] Furthermore, semaphorins can interfere with VEGF-mediated angiogenesis since both SEMA3s and VEGF-A compete for neuropilin receptor binding at endothelial cells.[36][37] The relative expression levels of SEMA3s and VEGF-A may therefore be important for angiogenesis.[18]

Chemical inhibition

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Main article: Angiogenesis inhibitor

An angiogenesis inhibitor can be endogenous or come from outside as drug or a dietary component.

Application in medicine

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Angiogenesis as a therapeutic target

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Angiogenesis may be a target for combating diseases such as heart disease characterized by either poor vascularisation or abnormal vasculature.[38] Application of specific compounds that may inhibit or induce the creation of new blood vessels in the body may help combat such diseases. The presence of blood vessels where there should be none may affect the mechanical properties of a tissue, increasing the likelihood of failure. The absence of blood vessels in a repairing or otherwise metabolically active tissue may inhibit repair or other essential functions. Several diseases, such as ischemic chronic wounds, are the result of failure or insufficient blood vessel formation and may be treated by a local expansion of blood vessels, thus bringing new nutrients to the site, facilitating repair. Other diseases, such as age-related macular degeneration, may be created by a local expansion of blood vessels, interfering with normal physiological processes.

The modern clinical application of the principle of angiogenesis can be divided into two main areas: anti-angiogenic therapies, which angiogenic research began with, and pro-angiogenic therapies. Whereas anti-angiogenic therapies are being employed to fight cancer and malignancies,[39][40] which require an abundance of oxygen and nutrients to proliferate, pro-angiogenic therapies are being explored as options to treat cardiovascular diseases, the number one cause of death in the Western world. One of the first applications of pro-angiogenic methods in humans was a German trial using fibroblast growth factor 1 (FGF-1) for the treatment of coronary artery disease.[23][41][42][43]

Regarding the mechanism of action, pro-angiogenic methods can be differentiated into three main categories: gene therapy, targeting genes of interest for amplification or inhibition; protein replacement therapy, which primarily manipulates angiogenic growth factors like FGF-1 or vascular endothelial growth factor, VEGF; and cell-based therapies, which involve the implantation of specific cell types.

There are still serious, unsolved problems related to gene therapy. Difficulties include effective integration of the therapeutic genes into the genome of target cells, reducing the risk of an undesired immune response, potential toxicity, immunogenicity, inflammatory responses, and oncogenesis related to the viral vectors used in implanting genes and the sheer complexity of the genetic basis of angiogenesis. The most commonly occurring disorders in humans, such as heart disease, high blood pressure, diabetes and Alzheimer's disease, are most likely caused by the combined effects of variations in many genes, and, thus, injecting a single gene may not be significantly beneficial in such diseases.[citation needed]

By contrast, pro-angiogenic protein therapy uses well-defined, precisely structured proteins, with previously defined optimal doses of the individual protein for disease states, and with well-known biological effects.[1] On the other hand, an obstacle of protein therapy is the mode of delivery. Oral, intravenous, intra-arterial, or intramuscular routes of protein administration are not always as effective, as the therapeutic protein may be metabolized or cleared before it can enter the target tissue. Cell-based pro-angiogenic therapies are still early stages of research, with many open questions regarding best cell types and dosages to use.

Tumor angiogenesis

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Without angiogenesis a tumor cannot grow beyond a limited size

Cancer cells are cells that have lost their ability to divide in a controlled fashion. A malignant tumor consists of a population of rapidly dividing and growing cancer cells that progressively accrues mutations. However, tumors need a dedicated blood supply to provide the oxygen and other essential nutrients they require in order to grow beyond a certain size (generally 1–2 mm3).[44][45]

Tumors induce blood vessel growth (angiogenesis) by secreting various growth factors (e.g. VEGF) and proteins. Growth factors such as bFGF and VEGF can induce capillary growth into the tumor, which some researchers suspect supply required nutrients, allowing for tumor expansion. Unlike normal blood vessels, tumor blood vessels are dilated with an irregular shape.[46] Other clinicians believe angiogenesis really serves as a waste pathway, taking away the biological end products secreted by rapidly dividing cancer cells. In either case, angiogenesis is a necessary and required step for transition from a small harmless cluster of cells, often said to be about the size of the metal ball at the end of a ball-point pen, to a large tumor. Angiogenesis is also required for the spread of a tumor, or metastasis.[9] Single cancer cells can break away from an established solid tumor, enter the blood vessel, and be carried to a distant site, where they can implant and begin the growth of a secondary tumor. Evidence now suggests the blood vessel in a given solid tumor may, in fact, be mosaic vessels, composed of endothelial cells and tumor cells.[9] This mosaicity allows for substantial shedding of tumor cells into the vasculature, possibly contributing to the appearance of circulating tumor cells in the peripheral blood of patients with malignancies.[47] The subsequent growth of such metastases will also require a supply of nutrients and oxygen and a waste disposal pathway.

Endothelial cells have long been considered genetically more stable than cancer cells. This genomic stability confers an advantage to targeting endothelial cells using antiangiogenic therapy, compared to chemotherapy directed at cancer cells, which rapidly mutate and acquire drug resistance to treatment. For this reason, endothelial cells are thought to be an ideal target for therapies directed against them.[48]

Formation of tumor blood vessels

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The mechanism of blood vessel formation by angiogenesis is initiated by the spontaneous dividing of tumor cells due to a mutation. Angiogenic stimulators are then released by the tumor cells. These then travel to already established, nearby blood vessels and activates their endothelial cell receptors. This induces a release of proteolytic enzymes from the vasculature. These enzymes target a particular point on the blood vessel and cause a pore to form. This is the point where the new blood vessel will grow from. The reason tumour cells need a blood supply is because they cannot grow any more than 2-3 millimeters in diameter without an established blood supply which is equivalent to about 50-100 cells.[49] Certain studies have indicated that vessels formed inside the tumor tissue are of higher irregularity and bigger in size, which is as well associated with poorer prognosis.[50][51]

Angiogenesis for cardiovascular disease

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Angiogenesis represents an excellent therapeutic target for the treatment of cardiovascular disease. It is a potent, physiological process that underlies the natural manner in which our bodies respond to a diminution of blood supply to vital organs, namely neoangiogenesis: the production of new collateral vessels to overcome the ischemic insult.[23] A large number of preclinical studies have been performed with protein-, gene- and cell-based therapies in animal models of cardiac ischemia, as well as models of peripheral artery disease. Reproducible and credible successes in these early animal studies led to high enthusiasm that this new therapeutic approach could be rapidly translated to a clinical benefit for millions of patients in the Western world with these disorders. A decade of clinical testing both gene- and protein-based therapies designed to stimulate angiogenesis in underperfused tissues and organs, however, has led from one disappointment to another. Although all of these preclinical readouts, which offered great promise for the transition of angiogenesis therapy from animals to humans, were in one fashion or another, incorporated into early stage clinical trials, the FDA has, to date (2007), insisted that the primary endpoint for approval of an angiogenic agent must be an improvement in exercise performance of treated patients.[52]

These failures suggested that either these are the wrong molecular targets to induce neovascularization, that they can only be effectively used if formulated and administered correctly, or that their presentation in the context of the overall cellular microenvironment may play a vital role in their utility. It may be necessary to present these proteins in a way that mimics natural signaling events, including the concentration, spatial and temporal profiles, and their simultaneous or sequential presentation with other appropriate factors.[53]

Exercise

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Angiogenesis is generally associated with aerobic exercise and endurance exercise. While arteriogenesis produces network changes that allow for a large increase in the amount of total flow in a network, angiogenesis causes changes that allow for greater nutrient delivery over a long period of time. Capillaries are designed to provide maximum nutrient delivery efficiency, so an increase in the number of capillaries allows the network to deliver more nutrients in the same amount of time. A greater number of capillaries also allows for greater oxygen exchange in the network. This is vitally important to endurance training, because it allows a person to continue training for an extended period of time. However, no experimental evidence suggests that increased capillarity is required in endurance exercise to increase the maximum oxygen delivery.[16]

Macular degeneration

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Overexpression of VEGF causes increased permeability in blood vessels in addition to stimulating angiogenesis. In wet macular degeneration, VEGF causes proliferation of capillaries into the retina. Since the increase in angiogenesis also causes edema, blood and other retinal fluids leak into the retina, causing loss of vision. Anti-angiogenic drugs targeting the VEGF pathways are now used successfully to treat this type of macular degeneration

Tissue engineered constructs

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Angiogenesis of vessels from the host body into an implanted tissue engineered constructs is essential. Successful integration is often dependent on thorough vascularisation of the construct as it provides oxygen and nutrients and prevents necrosis in the central areas of the implant.[54] PDGF has been shown to stabilize vascularisation in collagen-glycosaminoglycan scaffolds.[55]

History

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The first report of angiogenesis can be traced back to the book A treatise on the blood, inflammation, and gun-shot wounds published in 1794, where Scottish anatomist John Hunter's research findings were compiled. In his study, Hunter observed the growth process of new blood vessels in rabbits. However, he did not coin the term "Angiogenesis," which is now widely used by scholars. Hunter also erroneously attributed the growth process of new blood vessels to the effect of an innate vital principle within the blood. The term "angiogenesis" is believed to have emerged not until the 1900s. The inception of modern angiogenesis research is marked by Judah Folkman's report on the pivotal role of angiogenesis in tumor growth.[10][56][57]

Quantification

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Quantifying vasculature parameters such as microvascular density has various complications due to preferential staining or limited representation of tissues by histological sections. Recent research has shown complete 3D reconstruction of tumor vascular structure and quantification of vessel structures in whole tumors in animal models.[58]

See also

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References

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Revisions and contributorsEdit on WikipediaRead on Wikipedia

Angiogenesis

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from Grokipedia
Angiogenesis is the physiological process through which new blood vessels form from pre-existing vasculature, enabling the growth of networks essential for delivering oxygen and nutrients to tissues. This process occurs throughout life, beginning during embryonic development and continuing in adulthood to support , tissue repair, , and adaptation to physiological demands such as exercise-induced changes in and cardiac tissue. In healthy contexts, angiogenesis maintains tissue by ensuring no cell is more than a few hundred micrometers from a , thus preventing hypoxia and supporting metabolic functions. Key mechanisms include sprouting angiogenesis, where endothelial tip cells migrate in response to gradients of (VEGF), followed by stalk and lumen formation guided by Delta-Notch signaling, as well as intussusceptive angiogenesis involving vessel splitting for rapid network expansion. Major regulators encompass pro-angiogenic factors like VEGF and basic fibroblast growth factor (bFGF), which promote endothelial and migration, balanced by inhibitors such as endostatin and thrombospondin-1 that induce and limit vessel growth. Pathologically, dysregulated angiogenesis contributes to diseases including cancer, where tumors induce an "angiogenic switch" to sustain growth and ; ocular disorders like age-related macular degeneration; and chronic inflammatory conditions such as . Therapeutic strategies exploit these pathways, with anti-angiogenic agents like —a targeting VEGF—approved by the FDA in 2004 for treating and later expanded to other malignancies and neovascular eye diseases. Recent advances include combining anti-angiogenic agents with immunotherapies to enhance efficacy in treating various cancers. Conversely, pro-angiogenic therapies, such as recombinant bFGF, aim to stimulate vessel growth in ischemic conditions like . Research in this field, pioneered by in the 1970s with his hypothesis linking tumor progression to angiogenesis, has grown exponentially, with over 5,200 articles published in 2009 and informing ongoing clinical trials.

Introduction

Definition and Basic Process

Angiogenesis is the physiological process through which new blood vessels form from pre-existing vasculature, primarily involving the proliferation, migration, and reorganization of endothelial cells to create tubular structures that integrate into the vascular network. This process is essential for expanding the circulatory system in response to tissue demands, contrasting with vasculogenesis, which involves de novo vessel formation from endothelial precursor cells known as angioblasts during embryonic development. Unlike vasculogenesis, angiogenesis relies on the remodeling and extension of established vessels rather than initial assembly from isolated precursors. The basic process of angiogenesis unfolds in a series of coordinated steps, beginning with the degradation of the surrounding existing capillaries. Endothelial cells release proteases to break down this barrier, allowing cells to protrude and initiate vessel sprouting. A specialized endothelial cell is then selected as the "tip cell," which extends to sense environmental cues and lead directional migration toward angiogenic stimuli, such as (VEGF), which serves as a primary initiator of this response. Behind the tip cell, "stalk cells" proliferate to elongate the sprout, forming a multicellular column that maintains connectivity with the parent vessel. Subsequently, lumen formation occurs as endothelial cells rearrange to create hollow tubes, involving intracellular fusion and matrix remodeling to establish a conduit for blood flow. The nascent vessels then undergo , where sprouts from different sites connect to form functional loops, enabling circulation. Finally, vessel maturation stabilizes the structure, with recruited to the endothelial tubes to deposit components and regulate , while cells contribute to vessel wall reinforcement, ensuring long-term integrity and contractility. Endothelial cells remain central throughout, forming the inner lining and responding to signals for both sprouting and stabilization.

Biological Importance

Angiogenesis plays a fundamental role in normal by enabling the delivery of oxygen and nutrients to tissues during development, growth, and repair processes. In embryonic development, it supports the expansion of metabolically active tissues by forming new blood vessels from existing ones, ensuring adequate vascularization proportional to metabolic demands. This process is crucial for , where new capillary networks facilitate the influx of immune cells, nutrients, and oxygen to promote tissue regeneration and formation. Furthermore, angiogenesis maintains tissue by dynamically adjusting vascular density in response to physiological changes, such as increased capillary formation in during exercise or in with weight gain. Dysregulation of angiogenesis leads to significant pathological consequences, with excessive vessel formation contributing to diseases like cancer, chronic inflammation, and retinopathy. In tumors, angiogenesis is essential for growth beyond a microscopic size, as it provides the necessary blood supply for nutrient delivery and waste removal, a concept first proposed by in 1971. Overexpression of pro-angiogenic factors like VEGF drives aberrant neovascularization in chronic inflammatory conditions such as and , exacerbating tissue damage. In diabetic retinopathy, excessive retinal angiogenesis results in leaky, fragile vessels that cause vision loss through hemorrhage and edema. Conversely, insufficient angiogenesis impairs recovery in ischemic conditions, such as , where limited vessel growth restricts oxygen supply to hypoxic tissues, and delays in chronic ulcers by hindering nutrient delivery. The process of angiogenesis exhibits remarkable evolutionary conservation across vertebrates, including jawless fish, and shares mechanistic roots with hypoxia-sensing pathways in . Recent studies using have revealed heterogeneity in endothelial cell responses, further elucidating adaptive vascular remodeling (as of 2023). This conservation underscores its fundamental role in vascular adaptation across species and holds promise for , where harnessing conserved pathways could enhance tissue repair in humans. Angiogenesis operates in balance with vascular regression, forming a dynamic remodeling system where unused vessels regress—such as in muscle after disuse—while new ones form in response to stimuli, ensuring efficient resource allocation.

Types of Angiogenesis

Sprouting Angiogenesis

Sprouting angiogenesis represents the primary mechanism by which new blood vessels form from pre-existing capillaries, involving the directed outgrowth of endothelial cells to expand vascular networks. This process begins with the activation of endothelial cells in response to angiogenic stimuli, leading to the degradation of the and the (ECM) via matrix metalloproteinases and other proteases. The selected leading endothelial cells, known as tip cells, extend dynamic to sense and migrate toward pro-angiogenic cues, such as (VEGF) gradients, while trailing stalk cells proliferate to elongate the sprout. Central to sprout initiation is the selection of tip cells through lateral inhibition mediated by Delta-Notch signaling. Endothelial cells compete for tip cell fate; those with higher VEGF receptor 2 (VEGFR2) expression upregulate Delta-like 4 (Dll4), activating Notch in neighboring cells to suppress their tip cell characteristics and promote stalk cell identity instead. This results in a patterned arrangement of alternating tip and stalk cells, ensuring organized branching and preventing excessive sprout density. Delta-Notch interactions thus maintain a balance between migration at the sprout tip and proliferation in the stalk, with disruptions leading to hyper- or hypo-branching phenotypes observed in developmental models. As tip cells advance, they invade the surrounding ECM using filopodia protrusions stabilized by dynamics and , guiding the sprout through the tissue. Stalk cells follow, forming a primitive tubular structure via lumenogenesis, where intracellular vacuoles fuse to create a hollow vessel lumen. These primitive sprouts eventually anastomose—connecting tip cells from adjacent sprouts—to form functional vascular loops that enable flow and . are recruited to stabilize the nascent vessels, depositing new components to mature the network. This mode of angiogenesis predominates in embryonic vascular development, where it establishes the primary through patterned in structures like the and intersomitic vessels. It is also critical during , facilitating rapid neovascularization to supply oxygen and nutrients to repairing tissues. In pathological contexts, such as solid tumors, angiogenesis drives aberrant vessel growth, supporting tumor expansion by providing metabolic support despite often resulting in leaky, tortuous vessels.

Intussusceptive Angiogenesis

Intussusceptive angiogenesis, also known as splitting angiogenesis, is a mode of vessel formation characterized by the internal division of existing without the need for endothelial cell or significant proliferation. This process was first morphologically identified in the developing rat lung, where small transluminal pillars were observed perforating capillary walls. Unlike angiogenesis, it relies on the remodeling of preexisting vascular structures to rapidly expand microvascular networks. The mechanism begins with the formation of intravascular tissue pillars that span the lumen of a , typically initiated by points of contact between opposing endothelial cells. These pillars, often 1-1.5 μm in diameter, arise in regions of altered , such as low zones, and are influenced by mechanical forces like blood flow convergence at vessel bifurcations. The process unfolds in distinct phases: initial endothelial cell contact and protrusion formation, followed by perforation of the to create a transluminal bridge, involvement of pericytes to stabilize the structure, and finally deposition of components like for pillar maturation. As pillars grow and align in rows, they fuse into that bisect the vessel, leading to bifurcation and the creation of two parallel daughter vessels from a single parent , all without requiring extensive degradation or endothelial migration. Structurally, intussusceptive angiogenesis results in a rapid increase in capillary density through this internal partitioning, transforming two-dimensional networks into more complex three-dimensional architectures. The process is often reversible, allowing for pillar regression and vessel pruning to optimize network efficiency in response to changing demands. For instance, in models of adaptation, it can elevate the capillary-to-fiber ratio by 15-20%, enhancing tissue oxygenation without net cell addition. This form of angiogenesis is prevalent in contexts requiring swift vascular adaptation, such as alveolarization during postnatal development, where it facilitates a 20- to 30-fold increase in capillary volume from birth to adulthood in rats. It also occurs in tumor microenvironments adapting to hypoxia, enabling rapid vessel duplication within hours to days, and in inflammatory conditions like murine , where it supports tissue perfusion during acute responses. Shear stress variations, such as those from altered blood flow, can trigger pillar initiation in these settings. Compared to sprouting angiogenesis, intussusceptive angiogenesis offers key advantages in speed and efficiency, completing vessel splitting within hours or even minutes rather than days, and demanding minimal energy since it bypasses the need for endothelial proliferation and major extracellular remodeling. This makes it particularly suited for scenarios of rapid tissue expansion or adaptation under physiological constraints.

Coalescent Angiogenesis

Coalescent angiogenesis represents a distinct mode of vascular development characterized by the fusion of existing segments to form larger conduits, thereby optimizing blood flow efficiency without relying on from pre-existing vessels. This process involves the longitudinal of two or more smaller vessels, which merge along their axes to create a single, wider vessel capable of handling increased hemodynamic demands. Triggered primarily by detected through endothelial mechanoreceptors, the mechanism entails dynamic remodeling where endothelial cells align and fuse, often modulated by signaling pathways such as VEGF-induced Delta-like ligand 4 expression or Notch inhibition to facilitate cell-cell and lumen expansion. A key aspect of coalescent angiogenesis is the concurrent regression of underperfused or unnecessary vessels, which accompanies fusion to sculpt a hierarchical vascular network from an initial isotropic . This remodeling reduces the total number of vessels while increasing their diameters, transforming a low-resistance, inefficient into a tree-like structure that supports convective transport of nutrients and oxygen. Structural adaptations include the elimination of internal tissue pillars within fused segments, ensuring seamless integration and preventing flow disruptions, as evidenced by intravital studies in embryonic models showing phased progression from mesh formation to stabilized conduits over hours to days. This type of angiogenesis plays a critical role in embryonic vascular patterning, where it contributes to the maturation of major arteries like the dorsal aorta through symmetric fusion of primitive vessels in avian and mammalian models. In retinal development, coalescent processes aid in refining the superficial vascular plexus by merging nascent capillaries into deeper, more robust networks during early postnatal stages. Guidance by pro-maturational factors such as angiopoietins supports these fusions, promoting vessel stability during network reorganization.81426-9) Following fusion, recruitment is essential for stabilizing the newly formed larger vessels, where these mural cells invest along the to enhance structural integrity and regulate permeability. This post-fusion stabilization prevents regression of the remodeled conduits and ensures long-term functionality in the hierarchical network, as respond to PDGF-B signaling from endothelial cells to migrate and envelop the fused segments.

Regulation of Angiogenesis

Mechanical Factors

Mechanical forces play a pivotal role in regulating angiogenesis by influencing endothelial cell behavior and vascular network architecture independent of chemical mediators. These forces arise from hemodynamic conditions, extracellular matrix (ECM) interactions, and tissue deformations, guiding processes such as sprouting initiation and vessel stabilization. Key mechanical cues include hemodynamic shear stress from blood flow, interstitial flow through tissues, and tensile forces within the ECM, each modulating endothelial responses through mechanotransduction pathways. Hemodynamic , generated by blood flow along vessel walls, promotes angiogenic branching at low magnitudes (less than 10 dyn/cm²) while inhibiting excessive at physiological levels (10–70 dyn/cm²) to ensure vessel maturation and alignment. Interstitial flow, occurring at velocities up to 2 µm/s in perivascular spaces, directs endothelial via durotaxis along gradients and enhances tip cell polarization, facilitating sprout elongation and . Tensile forces, often from cyclic stretching of the ECM (5–15% strain), increase cell traction and cytoskeleton remodeling, thereby boosting migration and capillary on matrices with in the range of 500–2500 Pa. In contexts such as exercise-induced blood flow, these forces drive adaptive vascular remodeling in ; elevated shear and stretch in contribute to aberrant vessel thickening; and injury-related strain triggers matrix stiffening to support neovascularization. Endothelial cells sense these mechanical stimuli through mechanotransduction complexes involving , , and . (e.g., αvβ3 and α5β1) link the ECM to the , activating focal adhesion kinase (FAK) and Rho-associated kinase (ROCK) pathways to upregulate migration and proliferation in response to matrix tension and . , localized at cell-cell junctions, transmits signals via 3-kinase (PI3K)/Akt activation, promoting cell survival and directed sprouting. mediates stretch-induced junctional remodeling, weakening adherens junctions under high tension to enable tip cell specification and collective migration during branching. These pathways allow mechanics to amplify underlying chemical signals, such as enhancing (VEGF) responsiveness in one coordinated process.

Pro-angiogenic Chemical Signals

Pro-angiogenic chemical signals encompass a diverse array of soluble factors and matrix-associated proteins that orchestrate endothelial cell activation, migration, proliferation, and vessel maturation during angiogenesis. These molecules are primarily secreted by hypoxic tissues, inflammatory cells, and endothelial cells themselves, responding to cues like tissue injury or growth demands. Key families include vascular endothelial growth factors (VEGFs), fibroblast growth factors (FGFs), angiopoietins, matrix metalloproteinases (MMPs), and select signaling pathways such as Dll4-Notch and class 3 semaphorins, alongside platelet-derived growth factors (PDGFs), each contributing distinct mechanisms to vascular expansion. The VEGF family stands as the cornerstone of pro-angiogenic signaling, with VEGF-A being the predominant isoform that binds primarily to receptors (VEGFRs) 1 and 2 on endothelial cells. VEGF-A exists in multiple isoforms generated by , such as VEGF-A_{165} and VEGF-A_{121}, which differ in heparin-binding domains affecting their and . Upon binding to VEGFR-2, a receptor, VEGF-A triggers downstream pathways like PI3K/Akt and MAPK/ERK, promoting endothelial proliferation, migration, , and increased essential for sprout invasion into the (ECM). VEGFR-1 modulates these effects by sequestering VEGF-A or facilitating fine-tuned signaling, while VEGF-C and VEGF-D, though more lymphangiogenic, support angiogenesis via VEGFR-2 under certain conditions. Hypoxia-inducible factor (HIF)-1α upregulates VEGF expression in low-oxygen environments, initiating angiogenic cascades. Fibroblast growth factors, particularly FGF-1 and FGF-2 (basic FGF), exert potent mitogenic effects on endothelial cells by binding to fibroblast growth factor receptors (FGFRs), which are tyrosine kinases expressed on vascular . FGF-2, often released from damaged ECM or cells, activates FGFR-1 and FGFR-2, stimulating and MAPK pathways that enhance endothelial proliferation, migration, and production for ECM remodeling. These factors synergize with VEGFs to amplify angiogenesis, as evidenced in models of and tumor vascularization where FGF blockade impairs vessel formation. Unlike VEGFs, FGFs also recruit and smooth muscle cells to stabilize nascent vessels. Angiopoietins, including Ang-1 and Ang-2, modulate vessel maturation and remodeling through the Tie2 on endothelial cells. Ang-1, produced by and cells, acts as a Tie2 that promotes endothelial junction integrity, suppresses permeability, and recruits mural cells to stabilize mature vessels post-sprouting. In contrast, Ang-2, stored in Weibel-Palade bodies of endothelial cells and released upon stimulation, antagonizes Ang-1 at Tie2, destabilizing junctions to enable remodeling and responsiveness to pro-angiogenic cues like VEGF. This dynamic balance ensures appropriate vessel branching and regression during physiological angiogenesis. Matrix metalloproteinases, such as MMP-2 (gelatinase A) and MMP-9 (gelatinase B), facilitate angiogenesis by degrading ECM components like collagen IV and , creating paths for endothelial migration and sprout elongation. These zinc-dependent endopeptidases are secreted as pro-enzymes and activated by or other MMPs, with endothelial cells and inflammatory infiltrates as primary sources. MMP-2 and MMP-9 not only liberate ECM-bound growth factors like VEGF but also expose cryptic pro-angiogenic sites on matrix proteins, enhancing endothelial invasion without excessive tissue disruption. Among other regulators, the Dll4-Notch signaling pathway refines angiogenic branching by in endothelial tip cells. Dll4, a membrane-bound upregulated by VEGF on leading tip cells, activates Notch receptors on adjacent stalk cells, suppressing their responsiveness to VEGF and limiting excessive to maintain organized vessel patterns. Class 3 semaphorins, such as Sema3A and Sema3C, provide guidance cues during vascular patterning by interacting with neuropilin-1 and plexin receptors, promoting directed endothelial migration and fine-tuning branch orientation in developmental and reparative angiogenesis. (PDGF), particularly PDGF-BB, supports recruitment and vessel maturation by binding PDGF receptor-β on , indirectly enhancing endothelial stability and preventing regression of new vessels.

Anti-angiogenic Chemical Signals

Anti-angiogenic chemical signals are essential endogenous molecules that counteract pro-angiogenic factors to regulate vascular and prevent excessive formation. These inhibitors maintain a delicate balance during physiological processes such as embryonic development, where uncontrolled angiogenesis could lead to malformed vasculature, and in adulthood, where they suppress aberrant vessel growth in pathological conditions like tumor progression. Among the key endogenous inhibitors, thrombospondin-1 (TSP-1), a large , plays a pivotal role by binding to the receptor on endothelial cells, thereby activating signaling pathways that induce and inhibit and proliferation. Originally identified as the first natural , TSP-1 is secreted by various cell types including endothelial cells and platelets, and its expression is upregulated in response to tissue remodeling needs. Endostatin, a 20-kDa fragment derived from the C-terminal noncollagenous domain (NC1) of XVIII, potently suppresses endothelial cell proliferation, migration, and survival by disrupting -mediated signaling, particularly through α5β1 , and interfering with (VEGF) pathways. This inhibitor is generated via proteolytic cleavage by enzymes such as cathepsin L and is present in the of various tissues, contributing to the suppression of neovascularization in normal .00005-0) Similarly, angiostatin, a 38-kDa internal fragment cleaved from plasminogen by urokinase-type , inhibits endothelial cell and migration by binding to αvβ3 and F1F0 on the cell surface, thereby blocking ATP production necessary for angiogenic responses. Circulating in plasma as part of the fibrinolytic , angiostatin helps regulate vessel remodeling during and prevents pathological overgrowth. Soluble receptor-1 (sVEGFR-1, also known as sFlt-1) acts as a receptor that sequesters VEGF and (PlGF) in the extracellular space, preventing their interaction with membrane-bound VEGFR-2 on endothelial cells and thus dampening pro-angiogenic signaling. Produced by of the FLT1 gene in endothelial cells, monocytes, and trophoblasts, sFlt-1 levels increase under hypoxic conditions to fine-tune VEGF . Other notable inhibitors include interferon-alpha (IFN-α), a that downregulates the expression of pro-angiogenic factors such as basic fibroblast growth factor (bFGF), interleukin-8 (IL-8), and matrix metalloproteinase-9 (MMP-9) in endothelial and tumor cells, thereby suppressing vessel sprouting. Interleukin-4 (IL-4), an immune-modulatory , inhibits bFGF-induced endothelial proliferation and tube formation, often through upregulation of anti-angiogenic genes. Tissue inhibitors of metalloproteinases (TIMPs), particularly TIMP-2 and TIMP-3, block matrix degradation required for endothelial invasion; for instance, TIMP-2 binds α3β1 to halt independently of its MMP-inhibitory function, while TIMP-3 directly antagonizes VEGF binding to VEGFR-2. These anti-angiogenic signals operate through feedback mechanisms that sense local cues like hypoxia or tissue stress, upregulating inhibitor production to curb excessive vessel growth; for example, in embryonic development, TSP-1 and endostatin limit vascular overexpansion to ensure proper organ patterning, while in , their downregulation allows unchecked angiogenesis in tumors. This loop, often involving proteolytic generation of inhibitors from larger precursors, maintains vascular quiescence and prevents disorders arising from imbalanced angiogenesis.

Physiological Roles

Embryonic Development and Growth

Angiogenesis is essential for embryonic development, enabling the expansion and patterning of the vascular system to support tissue growth and organogenesis. In mouse embryos, the process initiates shortly after implantation, with the first signs of vascular development appearing in the extraembryonic yolk sac around embryonic day 7.5 (E7.5), where endothelial precursors undergo vasculogenesis to form blood islands. By E8.5, these coalesce into a primitive capillary plexus, marking the onset of angiogenic remodeling that refines the network into larger vessels. This early yolk sac angiogenesis is critical for nutrient exchange and provides a scaffold for intraembryonic vascular extension. The transition from vasculogenesis to angiogenesis occurs progressively from E8.5 onward, involving the sprouting of new vessels from the initial plexus to establish a hierarchical circulatory system. This includes arterial-venous specification, where endothelial cells differentiate into artery- or vein-specific subtypes based on cues like blood flow hemodynamics and molecular signals such as ephrin-B2 and Coup-TFII, beginning around E9.5 in the yolk sac and embryo proper. Neural guidance cues, including semaphorins (e.g., Sema3A) and netrins, play a pivotal role in this patterning by directing endothelial tip cell migration and filopodial extension, ensuring precise vascular alignment with developing tissues. Sprouting angiogenesis, the dominant mechanism here, relies on VEGF gradients to drive tip cell selection and stalk cell proliferation. Organ-specific vascularization exemplifies angiogenesis's role in embryogenesis. In the , vessels sprout from a perineural around E9.0-E10.5, invading the under VEGF and Wnt signaling to form a ramified network and initiate blood- barrier formation by E12.5. Coronary angiogenesis in the heart begins at E11.5, with endothelial cells from and sprouting into the myocardium to vascularize the compact layer; recent lineage tracing confirms contributions from multiple progenitors including and , with minor input from epicardial cells, dependent on factors like PDGF-B and supported by epicardial cues. In limb buds, initial vascular ingress occurs at E9.5 via angiogenic sprouts from the intersomitic vessels and dorsal , forming a primitive that patterns the limb's arterial arches and venous drainage, influenced by FGF and BMP gradients from the apical ectodermal ridge. Genetic models underscore the indispensability of angiogenesis regulators. Homozygous null mutations in VEGF lead to embryonic lethality between E8.5 and E9.5, characterized by arrested endothelial cell differentiation and failure to form vessels or embryonic . Similarly, Tie2 (Tek) knockout mice succumb at E9.5 with disorganized endothelial clusters in the and embryo, lacking proper vessel remodeling and integrity due to impaired signaling. These phenotypes highlight how disruptions in core pathways halt developmental progression, preventing organ vascularization and tissue viability.

Wound Healing and Tissue Repair

Angiogenesis plays a crucial role in by supplying oxygen and nutrients to the injured tissue, facilitating the repair process through the formation of new blood vessels from existing ones. In the inflammatory phase, signals from immune cells trigger angiogenesis, where endothelial cells from nearby vessels proliferate and migrate into the site to form sprouts that invade the fibrin-rich clot. This process is essential for establishing a provisional vascular network that supports subsequent repair stages. During the proliferative phase, these sprouts organize into a mature microvascular network within the , a fibrovascular matrix composed of fibroblasts, , and new vessels that fills the wound bed and promotes re-epithelialization. Key drivers include hypoxia in the wound bed, which stabilizes hypoxia-inducible factor-1α (HIF-1α) to upregulate pro-angiogenic factors like (VEGF), and macrophage-secreted factors such as VEGF and basic fibroblast growth factor (bFGF), which amplify endothelial cell proliferation and migration. In the remodeling phase, excess vessels undergo regression through , the network to restore normal tissue architecture and prevent excessive scarring. In chronic wounds, such as diabetic ulcers, angiogenesis is often impaired due to an excess of anti-angiogenic inhibitors like endostatin and thrombospondin-1, alongside reduced pro-angiogenic signaling from hyperglycemia-induced , leading to persistent hypoxia and stalled formation. This dysregulation results in non-healing ulcers that affect millions annually and increase risk. Conversely, regenerative potential is highlighted in scarless fetal , where angiogenesis is enhanced with a 2-fold increase in vessel density and elevated VEGF expression at early gestational stages, enabling rapid tissue regeneration without . Matrix metalloproteinases (MMPs) contribute to this by remodeling the to support endothelial invasion, as detailed in pro-angiogenic signaling pathways.

Exercise-Induced Vascular Adaptation

Physical exercise stimulates angiogenesis primarily in to meet heightened metabolic demands, enhancing tissue and oxygen delivery during activity. This adaptive response involves the formation of new capillaries through mechanisms such as and intussusceptive angiogenesis, driven by both hemodynamic and biochemical cues. In endurance-trained athletes, these changes result in a more efficient vascular network, supporting prolonged performance without excessive fatigue. The primary mechanisms triggering exercise-induced capillary growth include shear stress from increased blood flow and metabolic perturbations like hypoxia and lactate accumulation. , a mechanical force exerted on endothelial cells by elevated blood velocity during exercise, promotes angiogenesis by upregulating (NO) production and (VEGF) expression, as demonstrated in models where blocking VEGF abolished shear-dependent vessel formation. Concurrently, metabolic demand signals such as lactate—produced during anaerobic —act via the HCAR1 receptor to stabilize hypoxia-inducible factor-1α (HIF-1α), thereby enhancing VEGF from muscle fibers and fostering sprouting. These processes, while interconnected, differ from general mechanical factors in their exercise-specific integration of flow dynamics with tissue-level hypoxia. Key adaptations include an elevated -to-fiber ratio, which improves oxygen diffusion and nutrient supply to muscle cells. In untrained individuals, can increase this ratio by 10-20% within weeks, while in athletes, it may rise by up to 30%, correlating with enhanced aerobic capacity and reduced reliance on anaerobic pathways. This vascular remodeling optimizes oxygen delivery, as evidenced by higher VO₂max and capillary density in trained versus sedentary populations. At the molecular level, coactivator 1-alpha (PGC-1α) plays a central role by upregulating VEGF transcription in response to exercise-induced hypoxia, independent of HIF-1α in some contexts. PGC-1α , triggered by AMPK signaling during contraction, not only drives angiogenesis but also coordinates , ensuring matched vascular and metabolic adaptations; knockout studies show 60-80% reductions in VEGF protein levels and ~20% reductions in capillary-to-fiber ratios without PGC-1α. VEGF, released primarily from myofibers, binds endothelial receptors to initiate endothelial and migration. Despite these benefits, exercise-induced angiogenesis exhibits limits, including plateau effects where capillary growth stabilizes after initial phases, as seen in studies showing no further increases beyond 4-8 weeks of endurance exercise. Sustained is required to maintain these adaptations and prevent , with detraining leading to rapid capillary .

Pathological Implications

Tumor Angiogenesis and Vessel Formation

Tumors initially grow as avascular masses limited to approximately 1-2 mm in diameter, relying on for nutrient and oxygen supply, beyond which ensues without vascularization. This constraint prompts the "angiogenic switch," where tumor cells transition to an angiogenic , secreting pro-angiogenic factors to induce vessel formation and support further expansion. In many solid tumors, this process predominantly involves angiogenesis, adapting normal vascular mechanisms to the pathological tumor environment. Hypoxia within the expanding avascular tumor core activates hypoxia-inducible factor-1 (HIF-1), which transcriptionally upregulates (VEGF) expression in tumor cells. Secreted VEGF binds to endothelial receptors on nearby vessels, promoting endothelial , migration, and tube formation to generate new tumor vasculature. Concurrently, an imbalance favoring angiopoietin-2 (Ang-2) over angiopoietin-1 destabilizes existing vessels by disrupting pericyte-endothelial interactions, resulting in immature, leaky vessels that facilitate initial tumor but exhibit structural disarray. Tumor-induced vessels are characteristically abnormal, featuring tortuous, dilated, and irregularly branched structures with increased permeability due to fenestrations and discontinuous membranes. These irregularities lead to heterogeneous blood flow, regions of poor , and persistent intratumoral hypoxia, which paradoxically sustains further VEGF production and angiogenic drive. The hyperpermeable enables extravasation of plasma proteins and cells, creating a fibrin-rich matrix that supports tumor invasion and facilitates by providing routes for tumor cell intravasation. Vascular heterogeneity in tumors arises from inconsistent perivascular coverage, with often deficient or loosely associated, failing to stabilize vessels and contributing to their immaturity. This deficit exacerbates vessel leakiness and regression susceptibility, fostering a microenvironment that promotes tumor progression and resistance to physiological constraints on growth.

Ocular Diseases

Excessive angiogenesis plays a central role in several ocular diseases, leading to pathological vessel growth that disrupts normal retinal and choroidal function, ultimately causing vision impairment or blindness. In conditions such as wet age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR), aberrant neovascularization arises from imbalances in angiogenic signaling, primarily driven by (VEGF), which promotes endothelial and vessel permeability. These disorders highlight how local environmental stressors in the eye trigger uncontrolled vascularization, distinct from physiological angiogenesis due to the fragile and leaky nature of the new vessels. Wet , the neovascular form of age-related , is characterized by (CNV), where new blood vessels grow from the into the sub-retinal pigment epithelium space or the sub-retinal space. This process is initiated by local hypoxia in the aging and , compounded by , which upregulates VEGF expression from retinal pigment epithelial cells and other sources, driving endothelial migration and tube formation. The resulting vessels are immature and permeable, leading to fluid leakage, , hemorrhage, and that distort the and cause central vision loss. Progression typically begins with accumulation and outer creating hypoxic avascular zones, followed by invasive CNV that breaches and the blood- barrier, exacerbating leakage into the neurosensory . A unique aspect of CNV in wet is the disruption of the outer blood- barrier at , allowing choroidal vessels to invade the avascular outer , which is normally shielded from systemic circulation. Diabetic retinopathy, particularly its proliferative stage (PDR), involves neovascularization where fragile new vessels sprout from the or retinal veins into the vitreous or along the retinal surface. Hyperglycemia-induced retinal ischemia creates hypoxic areas of non-perfusion, triggering and the release of pro-angiogenic factors like VEGF from Müller glial cells and , which stimulate endothelial proliferation and vascular invasion. These vessels are prone to rupture, causing vitreous hemorrhage, tractional , and neovascular glaucoma, all contributing to severe vision loss. The disease progresses from initial microvasculopathy with capillary dropout forming avascular hypoxic zones to aggressive preretinal or intravitreal neovascularization that breaches the inner blood- barrier, allowing plasma leakage and inflammatory cell infiltration into the neural . Distinctively, the inner blood-retinal barrier's tight junctions in PDR are compromised by VEGF-mediated downregulation of and claudins, facilitating pathological vessel growth into normally avascular vitreous spaces. VEGF, as a dominant pro-angiogenic signal, underscores these mechanisms across both conditions.

Cardiovascular Disorders

In cardiovascular disorders, angiogenesis is often dysregulated, manifesting as either insufficient vessel formation in ischemic conditions or excessive, aberrant neovascularization that exacerbates disease progression. This imbalance contributes to poor tissue , plaque instability, and adverse cardiac remodeling, highlighting the critical role of angiogenic processes in maintaining vascular . In ischemic contexts, such as post-myocardial (MI), insufficient angiogenesis severely limits recovery by failing to restore adequate blood supply to the infarcted myocardium. Following acute MI, the hypoxic environment triggers angiogenic responses starting from the peri-infarcted border zone, but inadequate neovascularization leads to persistent ischemia, increased infarct size, and reduced cardiomyocyte survival. Mechanisms include disrupted signaling pathways like HIF-1α/VEGF activation and impaired endothelial cell proliferation due to excessive (ROS) or dysregulated microRNAs (e.g., miR-19a-3p inhibiting VEGF expression), which collectively hinder vessel maturation and . This deficiency promotes adverse left , characterized by and , ultimately progressing to chronic . Conversely, in , excessive plaque neovascularization promotes vessel instability through the formation of immature, leaky microvessels within the arterial wall. Hypoxia within advanced plaques induces angiogenesis primarily from adventitial , driven by HIF-1α and VEGF-A, resulting in fragile vessels that facilitate infiltration and intraplaque hemorrhage. These leaky structures extravasate red blood cells, leading to deposition, iron-mediated , and , which correlate strongly with plaque vulnerability (e.g., microvessel density r=0.99 with leakage). Ruptured plaques exhibit the highest neovessel density, often 2-3 times that of stable lesions, exacerbating accumulation and activity that precipitate acute events like . Underlying these dysregulations are key mechanisms such as impaired of endothelial progenitor cells (EPCs) and , which compromise angiogenic capacity across cardiovascular diseases. EPCs, essential for postnatal neovascularization and vascular repair, show reduced numbers and functionality in conditions like , , and hypercholesterolemia, correlating with endothelial injury and limited collateral formation. and risk factors disrupt EPC mobilization and integration into nascent vessels, while —marked by reduced bioavailability—further impairs EC migration and tube formation, perpetuating ischemia. Angiopoietins, for instance, modulate this process by stabilizing vessels, but their imbalance exacerbates dysfunction in ischemic settings. The outcomes of these angiogenic impairments often culminate in due to poor collateral vessel formation, which fails to compensate for chronic . In patients with stable and chronic total occlusion, factors like exposure inhibit angiogenesis by disrupting VEGFR2/PI3K/Akt signaling, reducing endothelial proliferation and recovery, and independently predicting inadequate collaterals (OR 1.043). This leads to diminished density in ischemic myocardium, worsening ventricular function and increasing mortality risk, as evidenced by lower limb ischemia models showing reduced blood flow with impaired growth. Overall, such deficiencies in collateral angiogenesis contribute to progressive by sustaining myocardial hypoxia and remodeling.

Therapeutic Applications

Promoting Angiogenesis

Promoting angiogenesis involves therapeutic strategies aimed at stimulating new formation to restore in ischemic tissues, particularly for conditions like (PAD) and myocardial ischemia. These approaches leverage angiogenic factors such as (VEGF) to enhance vascularization, addressing limitations in natural repair mechanisms. Clinical applications focus on delivering these factors through targeted methods to improve outcomes in tissue repair and regeneration. Gene therapy represents a key approach for promoting angiogenesis, primarily through VEGF delivery to upregulate local angiogenic signaling in ischemic regions. Adenoviral or plasmid-based vectors encoding VEGF-A have been used to stimulate endothelial , migration, and recruitment, showing efficacy in preclinical models of limb and cardiac ischemia. For instance, intramuscular VEGF transfer in PAD patients has demonstrated improved collateral vessel formation and limb in phase II trials. Protein administration offers a direct method to enhance angiogenesis by infusing recombinant VEGF or (FGF) proteins into affected areas. In PAD, intra-arterial VEGF-165 delivery has increased capillary density and walking distance in clinical studies, while in myocardial ischemia, intracoronary FGF-2 administration has promoted collateral growth and reduced symptoms. These therapies provide rapid onset but require repeated dosing due to short half-lives. Stem cell and endothelial (EPC) transplantation further augments angiogenesis by mobilizing cells that secrete angiogenic factors and integrate into nascent vessels. Autologous bone marrow-derived mononuclear cells or EPCs, when injected into ischemic limbs or hearts, enhance neovascularization and tissue in patients with critical limb ischemia and post-myocardial damage. A randomized trial of intramuscular EPC transplantation in severe limb ischemia reported significant healing and reduced rates at 24 weeks. In applications for PAD and myocardial ischemia, these strategies collectively improve blood flow and functional recovery; for example, combined and EPC delivery has shown synergistic effects in restoring hindlimb perfusion in animal models of . Wound healing augmentation benefits similarly, with VEGF protein or accelerating granulation tissue formation and epithelialization in chronic ulcers by boosting microvascular networks. Tissue engineering integrates these approaches using scaffolds embedded with growth factors like VEGF to support angiogenesis in and transplants. or decellularized scaffolds releasing controlled doses of angiogenic proteins promote vascular infiltration and maturation within engineered tissues, enabling viable organoid development for applications in skin grafts and vascularized implants. Preclinical studies demonstrate that VEGF-loaded scaffolds enhance vessel density in subcutaneously implanted , facilitating nutrient delivery for larger-scale tissue constructs. Despite these advances, challenges persist, including the transient effects of VEGF-based therapies, which often lead to short-lived vessel formation due to rapid protein degradation or immune clearance of vectors. Off-target growth poses additional risks, such as aberrant vessel leakage or from excessive VEGF signaling, contributing to inconsistent clinical outcomes. Recent progress, such as recombinant CCL28 protein administration, addresses some limitations by promoting stable angiogenesis via CCR10+ endothelial cells, improving cardiac repair and in models. Exercise serves as a natural promoter of angiogenesis through shear stress-induced VEGF expression, complementing therapeutic interventions in ischemic rehabilitation.

Inhibiting Angiogenesis

Anti-angiogenic therapies aim to suppress pathological neovascularization, particularly in cancers where tumor growth relies on sustained blood vessel formation. These approaches target key signaling pathways to starve tumors of nutrients and oxygen, thereby inhibiting proliferation and metastasis. Bevacizumab, a humanized monoclonal antibody that binds vascular endothelial growth factor A (VEGF-A), was the first approved angiogenesis inhibitor and is used in combination with chemotherapy for various solid tumors, including metastatic colorectal, non-small cell lung, and renal cell carcinomas. Sunitinib, an oral multitargeted tyrosine kinase inhibitor, blocks receptors such as VEGF receptors, platelet-derived growth factor receptors, and others involved in angiogenesis, demonstrating efficacy in advanced renal cell carcinoma and gastrointestinal stromal tumors by reducing tumor vascularization and progression. In ocular diseases, anti-angiogenic agents address aberrant vessel growth in conditions like neovascular age-related (). , a recombinant humanized fragment that neutralizes all isoforms of VEGF-A, is administered via intravitreal injection to inhibit , preserving in wet patients as shown in multicenter trials. Emerging strategies expand beyond traditional VEGF inhibition to enhance therapeutic durability. Immunotherapies modulating tumor-associated macrophages () reprogram these cells from pro-angiogenic M2 to anti-angiogenic M1 phenotypes, with 2024 advances in engineered macrophages demonstrating improved tumor infiltration and vascular suppression in preclinical models. Targets like delta-like ligand 4 (DLL4), a Notch pathway component, offer promise in VEGF-resistant tumors; DLL4 blockade disrupts tip cell selection in sprouting vessels, reducing tumor angiogenesis independently or synergistically with agents. Additionally, ivonescimab, a bispecific targeting PD-1 and VEGF-A, was approved in in May 2024 for advanced non-small cell in combination with , showing promise in ongoing global trials as of November 2025. Despite successes, challenges persist in clinical application. Resistance often arises through alternative pro-angiogenic pathways, such as or signaling, leading to tumor adaptation and relapse. Common side effects include due to systemic VEGF inhibition affecting normal vasculature, as well as proteinuria and increased risk. Some pro-angiogenic herbal compounds, like those from Chinese medicines, may inadvertently counter by enhancing vessel formation, though their clinical impact remains underexplored.

History

Early Discoveries

The earliest scientific insights into angiogenesis emerged in the 18th century through the work of Scottish surgeon John Hunter, who in the 1760s observed the dynamic growth of s in chick embryos, noting their proportionality to the metabolic demands of developing tissues. Hunter's observations underscored the adaptive nature of vascular expansion during rapid physiological processes, such as and embryonic development, laying foundational concepts for later research. The term "angiogenesis" was first used by John Hunter in 1787 to describe blood vessel growth in antlers. In the mid-19th century, advanced the cellular theory of , which laid groundwork for understanding its role in pathological processes including tissue remodeling and repair. Toward the end of the , Moritz Ribbert's 1880 experiments demonstrated that tumors actively induce neovascularization from adjacent host tissues, revealing a tumor-host interaction essential for growth. In 1907, Edwin Goldmann demonstrated, using transparent chamber models in rabbits, that tumors induce neovascularization from adjacent host tissues and that central tumor regions undergo due to limited vascular supply, underscoring the need for angiogenesis to support tumor growth beyond initial sizes. Early 20th-century tumor implantation studies reinforced these findings; for example, implants of tumor fragments in animal models, such as those conducted in the using ear chambers, illustrated that viable tumor growth is strictly dependent on rapid vascular ingrowth, without which tumors remained dormant or necrotic. In 1935, Arthur T. Hertig applied the term to de novo blood vessel formation in the developing of monkeys.

Key Milestones in Research

The molecular era of angiogenesis research began with Judah Folkman's seminal 1971 hypothesis, which posited that tumor growth is dependent on angiogenesis and that tumors secrete a factor to induce neovascularization, laying the groundwork for targeting this process therapeutically. This idea shifted the focus from tumor cells alone to the , predicting that inhibiting angiogenesis could restrict tumor expansion beyond microscopic sizes. A major breakthrough came in the 1980s with the isolation of vascular permeability factor (VPF) from tumor fluid in 1983 by Harold Dvorak and colleagues, a potent inducer of vascular leakage later recognized as a key angiogenic mediator. Building on this, between 1989 and 1990, Napoleone Ferrara's team at isolated and cloned (VEGF) from bovine pituitary cells and tumor cells, identifying it as a specific for endothelial cells and establishing VEGF as the primary driver of pathological angiogenesis. These discoveries enabled the molecular characterization of angiogenesis signaling pathways, transforming it from a descriptive phenomenon into a targetable process. In the , research expanded to endogenous inhibitors, with Michael O'Reilly in Folkman's laboratory isolating angiostatin in 1994 from the conditioned medium of a Lewis lung , revealing it as a kringle-domain fragment of plasminogen that selectively suppresses endothelial and . Shortly after, in 1997, the same group discovered endostatin, a XVIII fragment extracted from a tumor, which potently inhibits angiogenesis and tumor growth in preclinical models by disrupting endothelial cell survival and migration. These findings demonstrated that tumors produce their own angiogenesis inhibitors, explaining dormancy mechanisms and inspiring a new class of anti-angiogenic agents. During the same decade, Peter Burri and colleagues elucidated intussusceptive angiogenesis, a non- mode of vessel formation first observed in the developing rat lung in 1986 and mechanistically detailed in 1990, where existing capillaries divide internally via pillar formation to rapidly expand networks without endothelial proliferation. This process, distinct from traditional , was shown to contribute to adaptive vascular remodeling in physiological and pathological contexts, broadening the understanding of angiogenic diversity. Therapeutic translation advanced significantly with the 2004 FDA approval of (Avastin), the first anti-angiogenic drug, a targeting VEGF, which extended survival in metastatic when combined with in pivotal trials. This milestone validated Folkman's hypothesis clinically, establishing anti-VEGF therapy as a cornerstone for and spurring approvals in other cancers. In the 2010s and 2020s, single-cell RNA sequencing (scRNA-seq) has unveiled profound endothelial cell heterogeneity in angiogenesis, with seminal work by Kalucka et al. in 2020 mapping transcriptomic profiles across murine tissues and identifying distinct angiogenic subtypes responsive to stimuli like VEGF. These studies revealed context-specific endothelial populations, such as tip, stalk, and cells, with varying proliferative and migratory potentials, enhancing insights into therapeutic resistance and vascular normalization.

Measurement and Quantification

In Vitro and In Vivo Assays

In vitro assays provide controlled environments to study individual steps of angiogenesis, such as endothelial , migration, and differentiation, using isolated cells or simplified matrices. These models allow for of pro- and anti-angiogenic factors but simplify the complex multicellular interactions present . One widely adopted is the endothelial tube formation on , where human umbilical vein endothelial cells (HUVECs) or other endothelial cells are seeded onto a extract like , a gelled matrix derived from . Within 4-16 hours, the cells reorganize into capillary-like tube structures mimicking vascular lumen formation, enabling assessment of angiogenic potential in response to stimuli such as (VEGF). This is valued for its simplicity and reproducibility, with tube formation quantified by parameters including total branch length and number of branching nodes. The scratch wound migration assay evaluates endothelial cell motility, a key early step in angiogenesis. In this method, a confluent monolayer of endothelial cells is scratched with a sterile tool to create a denuded area, and cell migration into the wound is monitored over 24-48 hours using time-lapse imaging. Factors like VEGF can enhance closure rates, reflecting chemotactic responses. Migration speed and wound closure percentage serve as primary metrics. Ex vivo assays bridge simplicity and complexity by using intact tissue explants. The aortic ring sprouting assay involves embedding transverse sections of or aorta in a three-dimensional or matrix, where microvessels sprout outward over 7-14 days, recapitulating sprouting angiogenesis with contributions from and fibroblasts. Sprout length, vessel density, and invasion area are common metrics to gauge angiogenic activity. Another prominent ex vivo model is the chick chorioallantoic membrane (CAM) assay, utilizing the vascularized extra-embryonic membrane of 8-10 day old chicken embryos. Implants such as tumor fragments or pellets are placed on the CAM, inducing vessel invasion and remodeling observable over 3-5 days, providing insights into angiogenic responses in a developing vascular bed. Metrics include the area of vessel invasion and density around the implant. Across these assays, key quantitative metrics focus on structural features to assess angiogenic extent, such as cumulative branch length (total vessel elongation in micrometers), node count (number of junctions or endpoints indicating network complexity), and invasion area (spatial coverage of sprouts in square millimeters). These parameters are often analyzed using software like ImageJ's Angiogenesis Analyzer for automated, reproducible measurements. Despite their utility, and assays have limitations, primarily the absence of full physiological context including blood flow, immune interactions, and systemic hormonal influences, which can lead to discrepancies with in vivo outcomes. Additionally, variability in matrix composition and cell sourcing affects reproducibility, and these models often overlook long-term vessel maturation and stability.

Imaging and Molecular Techniques

Imaging techniques play a crucial role in visualizing and quantifying angiogenesis in vivo, enabling non-invasive assessment of vascular development and response to therapies. Magnetic resonance imaging (MRI) enhanced with gadolinium (Gd)-based probes has emerged as a powerful tool for monitoring anti-angiogenic effects, particularly in tumor models. Recent advances in 2025 introduced Gd-DOTA-G3CNGRC, a targeted probe that binds specifically to aminopeptidase N (APN/CD13) on angiogenic endothelial cells, allowing early detection of therapeutic efficacy through enhanced contrast in T1-weighted images. This probe demonstrated superior specificity compared to non-targeted Gd agents, with signal intensity reductions correlating to decreased vascular permeability post-treatment in preclinical studies. Intravital microscopy provides dynamic, real-time visualization of angiogenic processes at the cellular level, capturing , sprout formation, and vascular remodeling in living tissues. By employing multiphoton or confocal techniques, researchers can track multi-cellular interactions during angiogenesis, such as tip cell guidance and extension, with resolutions down to micrometers. This method has revealed flow-directed endothelial behaviors in models like the wounded , where serial imaging over days highlights temporal changes in vessel and maturation. Molecular techniques offer precise quantification of angiogenic activity through gene and protein expression . Quantitative polymerase chain reaction (qPCR) is widely used to measure mRNA levels of key angiogenic factors like (VEGF) and angiopoietins, providing insights into transcriptional regulation during hypoxia-induced angiogenesis. (ELISA) detects soluble markers such as circulating VEGF and (sFlt-1), which reflect systemic angiogenic states and therapeutic modulation in serum samples. Single-cell RNA sequencing (scRNA-seq) unveils endothelial cell heterogeneity in angiogenic niches, identifying subpopulations with distinct or profiles that drive vessel instability. In vivo models leverage species-specific advantages for imaging angiogenesis. The transparency of zebrafish larvae facilitates high-resolution optical imaging of subintestinal vessels, allowing non-invasive tracking of angiogenic sprouting in response to genetic or pharmacological perturbations without pigmentation interference. In mice, the hindlimb ischemia model induces robust angiogenesis via ligation, enabling longitudinal assessment of collateral vessel formation and recovery over weeks. Quantitative metrics derived from these techniques standardize angiogenesis evaluation. Microvessel density (MVD), often assessed via immunostaining, serves as a surrogate for angiogenic extent, with elevated counts indicating proliferative vascular networks in ischemic tissues. Perfusion rates, measured through dynamic contrast-enhanced imaging or laser Doppler flowmetry, quantify functional blood flow, revealing improvements in vessel patency post-angiogenic stimulation. Integration of (AI) in image analysis, as advanced in 2024 protocols, automates vessel segmentation and density calculations from data, enhancing and reducing manual bias in large datasets.

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