Intussusceptive Vascular Remodeling Precedes Pathological Neovascularization.

Objective- Pathological neovascularization is crucial for progression and morbidity of serious diseases such as cancer, diabetic retinopathy, and age-related macular degeneration. While mechanisms of ongoing pathological neovascularization have been extensively studied, the initiating pathological vascular remodeling (PVR) events, which precede neovascularization remains poorly understood. Here, we identify novel molecular and cellular mechanisms of preneovascular PVR, by using the adult choriocapillaris as a model. Approach and Results- Using hypoxia or forced overexpression of VEGF (vascular endothelial growth factor) in the subretinal space to induce PVR in zebrafish and rats respectively, and by analyzing choriocapillaris membranes adjacent to choroidal neovascular lesions from age-related macular degeneration patients, we show that the choriocapillaris undergo robust induction of vascular intussusception and permeability at preneovascular stages of PVR. This PVR response included endothelial cell proliferation, formation of endothelial luminal processes, extensive vesiculation and thickening of the endothelium, degradation of collagen fibers, and splitting of existing extravascular columns. RNA-sequencing established a role for endothelial tight junction disruption, cytoskeletal remodeling, vesicle- and cilium biogenesis in this process. Mechanistically, using genetic gain- and loss-of-function zebrafish models and analysis of primary human choriocapillaris endothelial cells, we determined that HIF (hypoxia-induced factor)-1α-VEGF-A-VEGFR2 signaling was important for hypoxia-induced PVR. Conclusions- Our findings reveal that PVR involving intussusception and splitting of extravascular columns, endothelial proliferation, vesiculation, fenestration, and thickening is induced before neovascularization, suggesting that identifying and targeting these processes may prevent development of advanced neovascular disease in the future. Visual Overview- An online visual overview is available for this article.

Pericyte-fibroblast transition promotes tumor growth and metastasis.

Vascular pericytes, an important cellular component in the tumor microenvironment, are often associated with tumor vasculatures, and their functions in cancer invasion and metastasis are poorly understood. Here we show that PDGF-BB induces pericyte-fibroblast transition (PFT), which significantly contributes to tumor invasion and metastasis. Gain- and loss-of-function experiments demonstrate that PDGF-BB-PDGFRß signaling promotes PFT both in vitro and in in vivo tumors. Genome-wide expression analysis indicates that PDGF-BB-activated pericytes acquire mesenchymal progenitor features. Pharmacological inhibition and genetic deletion of PDGFRß ablate the PDGF-BB-induced PFT. Genetic tracing of pericytes with two independent mouse strains, TN-AP-CreERT2:R26R-tdTomato and NG2-CreERT2:R26R-tdTomato, shows that PFT cells gain stromal fibroblast and myofibroblast markers in tumors. Importantly, coimplantation of PFT cells with less-invasive tumor cells in mice markedly promotes tumor dissemination and invasion, leading to an increased number of circulating tumor cells and metastasis. Our findings reveal a mechanism of vascular pericytes in PDGF-BB-promoted cancer invasion and metastasis by inducing PFT, and thus targeting PFT may offer a new treatment option of cancer metastasis.

Collaborative interplay between FGF-2 and VEGF-C promotes lymphangiogenesis and metastasis.

Interplay between various lymphangiogenic factors in promoting lymphangiogenesis and lymphatic metastasis remains poorly understood. Here we show that FGF-2 and VEGF-C, two lymphangiogenic factors, collaboratively promote angiogenesis and lymphangiogenesis in the tumor microenvironment, leading to widespread pulmonary and lymph-node metastases. Coimplantation of dual factors in the mouse cornea resulted in additive angiogenesis and lymphangiogenesis. At the molecular level, we showed that FGFR-1 expressed in lymphatic endothelial cells is a crucial receptor that mediates the FGF-2-induced lymphangiogenesis. Intriguingly, the VEGFR-3-mediated signaling was required for the lymphatic tip cell formation in both FGF-2- and VEGF-C-induced lymphangiogenesis. Consequently, a VEGFR-3-specific neutralizing antibody markedly inhibited FGF-2-induced lymphangiogenesis. Thus, the VEGFR-3-induced lymphatic endothelial cell tip cell formation is a prerequisite for FGF-2-stimulated lymphangiogenesis. In the tumor microenvironment, the reciprocal interplay between FGF-2 and VEGF-C collaboratively stimulated tumor growth, angiogenesis, intratumoral lymphangiogenesis, and metastasis. Thus, intervention and targeting of the FGF-2- and VEGF-C-induced angiogenic and lymphangiogenic synergism could be potentially important approaches for cancer therapy and prevention of metastasis.

VEGFR1-mediated pericyte ablation links VEGF and PlGF to cancer-associated retinopathy.

VEGF coordinates complex regulation of cellular regeneration and interactions between endothelial and perivascular cells; dysfunction of the VEGF signaling system leads to retinopathy. Here, we show that systemic delivery of VEGF and placental growth factor (PlGF) by protein implantation, tumors, and adenoviral vectors ablates pericytes from the mature retinal vasculature through the VEGF receptor 1 (VEGFR1)-mediated signaling pathway, leading to increased vascular leakage. In contrast, we demonstrate VEGF receptor 2 (VEGFR2) is primarily expressed in nonvascular photoreceptors and ganglion cells. Moreover, blockade of VEGFR1 but not VEGFR2 significantly restores pericyte saturation in mature retinal vessels. Our findings link VEGF and PlGF to cancer-associated retinopathy, reveal the molecular mechanisms of VEGFR1 ligand-mediated retinopathy, and define VEGFR1 as an important target of antiangiogenic therapy for treatment of retinopathy.

Arteriogenic therapy by intramyocardial sustained delivery of a novel growth factor combination prevents chronic heart failure.

BACKGROUND: Therapeutic angiogenesis is a promising approach for the treatment of cardiovascular diseases, including myocardial infarction and chronic heart failure. We aimed to improve proangiogenic therapies by identifying novel arteriogenic growth factor combinations, developing injectable delivery systems for spatiotemporally controlled growth factor release, and evaluating functional consequences of targeted intramyocardial growth factor delivery in chronic heart failure. METHODS AND RESULTS: First, we observed that fibroblast growth factor and hepatocyte growth factor synergistically stimulate vascular cell migration and proliferation in vitro. Using 2 in vivo angiogenesis assays (n=5 mice per group), we found that the growth factor combination results in a more potent and durable angiogenic response than either growth factor used alone. Furthermore, we determined that the molecular mechanisms involve potentiation of Akt and mitogen-activated protein kinase signal transduction pathways, as well as upregulation of angiogenic growth factor receptors. Next, we developed crosslinked albumin-alginate microcapsules that sequentially release fibroblast growth factor-2 and hepatocyte growth factor. Finally, in a rat model of chronic heart failure induced by coronary ligation (n=14 to 15 rats per group), we found that intramyocardial slow release of fibroblast growth factor-2 with hepatocyte growth factor potently stimulates angiogenesis and arteriogenesis and prevents cardiac hypertrophy and fibrosis, as determined by immunohistochemistry, leading to improved cardiac perfusion after 3 months, as shown by magnetic resonance imaging. These multiple beneficial effects resulted in reduced adverse cardiac remodeling and improved left ventricular function, as revealed by echocardiography. CONCLUSION: Our data showing the selective advantage of using fibroblast growth factor-2 together with hepatocyte growth factor suggest that this growth factor combination may constitute an efficient novel treatment for chronic heart failure.

Placenta growth factor-1 antagonizes VEGF-induced angiogenesis and tumor growth by the formation of functionally inactive PlGF-1/VEGF heterodimers.

Tumor growth and metastasis require concomitant growth of new blood vessels, which are stimulated by angiogenic factors, including vascular endothelial growth factor (VEGF), secreted by most tumors. Whereas the angiogenic property and molecular mechanisms of VEGF have been well studied, the biological function of its related homolog, placenta growth factor (PlGF), is poorly understood. Here we demonstrate that PlGF-1, an alternatively spliced isoform of the PlGF gene, antagonizes VEGF-induced angiogenesis when both factors are coexpressed in murine fibrosarcoma cells. Overexpression of PlGF-1 in VEGF-producing tumor cells results in the formation of PlGF-1/VEGF heterodimers and depletion of the majority of mouse VEGF homodimers. The heterodimeric form of PlGF-1/VEGF lacks the ability to induce angiogenesis in vitro and in vivo. Similarly, PlGF-1/VEGF fails to activate the VEGFR-2-mediated signaling pathways. Further, PlGF-1 inhibits the growth of a murine fibrosarcoma by approximately 90% when PlGF-1-expressing tumor cells are implanted in syngeneic mice. In contrast, overexpression of human VEGF in murine tumor cells causes accelerated and exponential growth of primary fibrosarcomas and early hepatic metastases. Our data demonstrate that PlGF-1, a member of the VEGF family, acts as a natural antagonist of VEGF when both factors are synthesized in the same population of cells. The underlying mechanism is due to the formation of functionally inactive heterodimers.

PDGF-BB induces intratumoral lymphangiogenesis and promotes lymphatic metastasis.

Cancer metastases are commonly found in the lymphatic system. Like tumor blood angiogenesis, stimulation of tumor lymphangiogenesis may require the interplay of several tumor-derived growth factors. Here we report that members of the PDGF family act as lymphangiogenic factors. In vitro, PDGF-BB stimulated MAP kinase activity and cell motility of isolated lymphatic endothelial cells. In vivo, PDGF-BB potently induced growth of lymphatic vessels. Expression of PDGF-BB in murine fibrosarcoma cells induced tumor lymphangiogenesis, leading to enhanced metastasis in lymph nodes. These data demonstrate that PDGF-BB is an important growth factor contributing to lymphatic metastasis. Thus, blockage of PDGF-induced lymphangiogenesis may provide a novel approach for prevention and treatment of lymphatic metastasis.

Malignant cell-derived PlGF promotes normalization and remodeling of the tumor vasculature.

Vascular functions of PlGF remain poorly understood and controversial. Here, we show that tumor cell-derived PlGF-1 and PlGF-2 displayed significant remodeling effects on the tumor vasculature, leading to a normalized vascular phenotype and improved functions against leakage. In two murine tumor models, that is, T241 fibrosarcoma and Lewis lung carcinoma, stable expression of PlGF-1 and PlGF-2 in tumor cells resulted in significant reduction of tumor microvascular density and branch formation. Markedly, the vasculature in PlGF-expressing tumors consisted of relatively large-diameter microvessels with substantial improvement of pericyte coverage. Similarly, PlGF-induced vascular normalization and remodeling were also observed in a spontaneous human choriocarcinoma that expressed endogenous PlGF. Our findings shed light on functions of PlGF as a vascular remodeling factor that normalizes the tumor vasculature and thus may have conceptual implications of cancer therapy.

Nitric oxide permits hypoxia-induced lymphatic perfusion by controlling arterial-lymphatic conduits in zebrafish and glass catfish.

The blood and lymphatic vasculatures are structurally and functionally coupled in controlling tissue perfusion, extracellular interstitial fluids, and immune surveillance. Little is known, however, about the molecular mechanisms that underlie the regulation of bloodlymphatic vessel connections and lymphatic perfusion. Here we show in the adult zebrafish and glass catfish (Kryptopterus bicirrhis) that blood-lymphatic conduits directly connect arterial vessels to the lymphatic system. Under hypoxic conditions, arterial-lymphatic conduits (ALCs) became highly dilated and linearized by NO-induced vascular relaxation, which led to blood perfusion into the lymphatic system. NO blockage almost completely abrogated hypoxia-induced ALC relaxation and lymphatic perfusion. These findings uncover mechanisms underlying hypoxia-induced oxygen compensation by perfusion of existing lymphatics in fish. Our results might also imply that the hypoxia-induced NO pathway contributes to development of progression of pathologies, including promotion of lymphatic metastasis by modulating arterial-lymphatic conduits, in the mammalian system.

Angiogenic synergism, vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2.

The establishment of functional and stable vascular networks is essential for angiogenic therapy. Here we report that a combination of two angiogenic factors, platelet-derived growth factor (PDGF)-BB and fibroblast growth factor (FGF)-2, synergistically induces vascular networks, which remain stable for more than a year even after depletion of angiogenic factors. In both rat and rabbit ischemic hind limb models, PDGF-BB and FGF-2 together markedly stimulated collateral arteriogenesis after ligation of the femoral artery, with a significant increase in vascularization and improvement in paw blood flow. A possible mechanism of angiogenic synergism between PDGF-BB and FGF-2 involves upregulation of the expression of PDGF receptor (PDGFR)-alpha and PDGFR-beta by FGF-2 in newly formed blood vessels. Our data show that a specific combination of angiogenic factors establishes functional and stable vascular networks, and provides guidance for the ongoing clinical trials of angiogenic factors for the treatment of ischemic diseases.

Anti-VEGF agents confer survival advantages to tumor-bearing mice by improving cancer-associated systemic syndrome.

The underlying mechanism by which anti-VEGF agents prolong cancer patient survival is poorly understood. We show that in a mouse tumor model, VEGF systemically impairs functions of multiple organs including those in the hematopoietic and endocrine systems, leading to early death. Anti-VEGF antibody, bevacizumab, and anti-VEGF receptor 2 (VEGFR-2), but not anti-VEGFR-1, reversed VEGF-induced cancer-associated systemic syndrome (CASS) and prevented death in tumor-bearing mice. Surprisingly, VEGFR2 blockage improved survival by rescuing mice from CASS without significantly compromising tumor growth, suggesting that "off-tumor" VEGF targets are more sensitive than the tumor vasculature to anti-VEGF drugs. Similarly, VEGF-induced CASS occurred in a spontaneous breast cancer mouse model overexpressing neu. Clinically, VEGF expression and CASS severity positively correlated in various human cancers. These findings define novel therapeutic targets of anti-VEGF agents and provide mechanistic insights into the action of this new class of clinically available anti-VEGF cancer drugs.

FOXC2 controls Ang-2 expression and modulates angiogenesis, vascular patterning, remodeling, and functions in adipose tissue.

Adipogenesis is spatiotemporally coupled to angiogenesis throughout adult life, and the interplay between these two processes is communicated by multiple factors. Here we show that in a transgenic mouse model, increased expression of forkhead box C2 (FOXC2) in the adipose tissue affects angiogenesis, vascular patterning, and functions. White and brown adipose tissues contain a considerably high density of microvessels appearing as vascular plexuses, which show redistribution of vascular smooth muscle cells and pericytes. Dysfunction of these primitive vessels is reflected by impairment of skin wound healing. We further provide a mechanistic insight of the vascular phenotype by showing that FOXC2 controls Ang-2 expression by direct activation of its promoter in adipocytes. Remarkably, an Ang-2-specific antagonist almost completely reverses this vascular phenotype. Thus, the FOXC2-Ang-2 signaling system is crucial for controlling adipose vascular function, which is part of an adaptation to increased adipose tissue metabolism.

Angiogenic factors FGF2 and PDGF-BB synergistically promote murine tumor neovascularization and metastasis.

Tumors produce multiple growth factors, but little is known about the interplay between various angiogenic factors in promoting tumor angiogenesis, growth, and metastasis. Here we show that 2 angiogenic factors frequently upregulated in tumors, PDGF-BB and FGF2, synergistically promote tumor angiogenesis and pulmonary metastasis. Simultaneous overexpression of PDGF-BB and FGF2 in murine fibrosarcomas led to the formation of high-density primitive vascular plexuses, which were poorly coated with pericytes and VSMCs. Surprisingly, overexpression of PDGF-BB alone in tumor cells resulted in dissociation of VSMCs from tumor vessels and decreased recruitment of pericytes. In the absence of FGF2, capillary ECs lacked response to PDGF-BB. However, FGF2 triggers PDGFR-alpha and -beta expression at the transcriptional level in ECs, which acquire hyperresponsiveness to PDGF-BB. Similarly, PDGF-BB-treated VSMCs become responsive to FGF2 stimulation via upregulation of FGF receptor 1 (FGFR1) promoter activity. These findings demonstrate that PDGF-BB and FGF2 reciprocally increase their EC and mural cell responses, leading to disorganized neovascularization and metastasis. Our data suggest that intervention of this non-VEGF reciprocal interaction loop for the tumor vasculature could be an important therapeutic target for the treatment of cancer and metastasis.

Differential roles of PDGFR-alpha and PDGFR-beta in angiogenesis and vessel stability.

Preclinical and clinical evaluations of individual proangiogenic/arteriogenic factors for the treatment of ischemic myocardium and skeletal muscle have produced unfulfilled promises. The establishment of functional and stable arterial vascular networks may require combinations of different angiogenic and arteriogenic factors. Using in vivo angiogenesis and ischemic hind-limb animal models, we have compared the angiogenic and therapeutic activities of fibroblast growth factor 2 (FGF-2) in combinations with PDGF-AA and PDGF-AB, two members of the platelet-derived growth factor (PDGF) family, with distinct receptor binding patterns. We show that both PDGF-AA/FGF-2 and PDGF-AB/FGF-2 in combinations synergistically induce angiogenesis in the mouse cornea. FGF-2 up-regulates PDGFR-alpha and -beta expression levels in the newly formed blood vessels. Interestingly, PDGF-AB/FGF-2, but not PDGF-AA/FGF-2, is able to stabilize the newly formed vasculature by recruiting pericytes, and an anti-PDGFR-beta neutralizing antibody significantly blocks PDGF-AB/FGF-2-induced vessel stability. These findings demonstrate that PDGFR-beta receptor is essential for vascular stability. Similarly, PDGF-AB/FGF-2 significantly induces stable collateral growth in the rat ischemic hind limb. The high number of collaterals induced by PDGF-AB/FGF-2 leads to dramatic improvement of the paw's skin perfusion. Immunohistochemical analysis of the treated skeletal muscles confirms that a combination of PDGF-AB and FGF-2 significantly induces arteriogenesis in the ischemic tissue. A combination of PDGF-AB and FGF-2 would be optimal proangiogenic agents for the treatment of ischemic diseases.

Cell-type-specific regulation of degradation of hypoxia-inducible factor 1 alpha: role of subcellular compartmentalization.

The hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a transcription factor that mediates adaptive cellular responses to decreased oxygen availability (hypoxia). At normoxia, HIF-1 alpha is targeted by the von Hippel-Lindau tumor suppressor protein (pVHL) for degradation by the ubiquitin-proteasome pathway. In the present study we have observed distinct cell-type-specific differences in the ability of various tested pVHL-interacting subfragments to stabilize HIF-1 alpha and unmask its function at normoxia. These properties correlated with differences in subcellular compartmentalization and degradation of HIF-1 alpha. We observed that the absence or presence of nuclear localization or export signals differently affected the ability of a minimal HIF-1 alpha peptide spanning residues 559 to 573 of mouse HIF-1 alpha to stabilize endogenous HIFalpha and induce HIF-driven reporter gene activity in two different cell types (primary mouse endothelial and HepG2 hepatoma cells). Degradation of HIF-1 alpha occurred mainly in the cytoplasm of HepG2 cells, whereas it occurs with equal efficiency in nuclear and cytoplasmic compartments of primary endothelial cells. Consistent with these observations, green fluorescent protein-HIF-1 alpha is differently distributed during hypoxia and reoxygenation in hepatoma and endothelial cells. Consequently, we propose that differential compartmentalization of degradation of HIF-1 alpha and the subcellular distribution of HIF-1 alpha may account for cell-type-specific differences in stabilizing HIF-1 alpha protein levels under hypoxic conditions.

R Regulation of tumor angiogenesis and metastasis by FGF and PDGF signaling pathways.

In a fast-growing malignant tissue, tumor blood vessels are exposed to multiple growth factors and cytokines. Although the role of individual factors and their signaling pathways in regulation of tumor neovascularization is relatively well-studied, little is known about complex interactions between these factors and their cooperative effects in promoting tumor angiogenesis and metastasis. Our recent studies show that quiescent vascular endothelial cells usually remaining silence to platelet-derived growth factor (PDGF)-BB stimulation acquire their hyperresponsiveness after stimulation with fibroblast growth factor (FGF)-2, which transcriptionally switches on PDGF receptor expression in the activated endothelial cells. Interestingly, PDGF-BB also transduces positive feedback signals to the FGF-2 signaling system by amplifying its receptor expression in vascular mural cells. These uncoordinated reciprocal interactions in the tumor environment lead to the formation of disorganized and primitive vasculatures that facilitate tumor growth and metastasis in mice. These findings provide an example of complex interaction between tumor angiogenic factors. Thus, therapeutic development of antiangiogenic agents for the treatment of cancer should be aimed to block multiple angiogenic signaling pathways and their interactive loops.

Deletion of the laminin alpha4 chain leads to impaired microvessel maturation.

The laminin alpha4 chain, a component of laminin-8 and -9, is expressed in basement membranes, such as those beneath endothelia, the perineurium of peripheral nerves, and around developing muscle fibers. Laminin alpha4-null mice presented with hemorrhages during the embryonic and neonatal period and had extensive bleeding and deterioration of microvessel growth in experimental angiogenesis, as well as mild locomotion defects. Histological examination of newborn mice revealed delayed deposition of type IV collagen and nidogen into capillary basement membranes, and electron microscopy showed discontinuities in the lamina densa. The results demonstrate a central role for the laminin alpha4 chain in microvessel growth and, in the absence of other laminin alpha chains, in the composition of endothelial basement membranes.

Vascular endothelial growth factor-a promotes peritumoral lymphangiogenesis and lymphatic metastasis.

Metastases are commonly found in the lymphatic system. The molecular mechanism of lymphatic metastasis is, however, poorly understood. Here we report that vascular endothelial growth factor (VEGF)-A stimulated lymphangiogenesis in vivo and that overexpression of VEGF-A in murine T241 fibrosarcomas induced the growth of peritumoral lymphatic vessels, which occasionally penetrated into the tumor tissue. As a result of peritumoral lymphangiogenesis, metastases in lymph nodes of mice were detected. VEGF-A-overexpressing tumors contained high numbers of infiltrating inflammatory cells such as macrophages, which are known to express VEGF receptor (VEGFR)-1. It seemed that in the mouse cornea, VEGF-A stimulated lymphangiogenesis through a VEGF-C/-D/VEGFR-3-independent pathway as a VEGFR-3 antagonist selectively inhibited VEGF-C-induced, but not VEGF-A-induced, lymphangiogenesis. Our data show that VEGF-A contributes to lymphatic mestastasis. Thus, blockage of VEGF-A-induced lymphangiogenesis may provide a novel approach for prevention and treatment of lymphatic metastasis.

Blockage of VEGF-induced angiogenesis by preventing VEGF secretion.

Vascular endothelial growth factor (VEGF)/vascular permeability factor is one of the most frequently expressed angiogenic factors in several pathological tissues. Development of VEGF antagonists has become an important approach in the treatment of angiogenesis-dependent diseases. Here we describe a novel anti-VEGF strategy by preventing the secretion of VEGF. We utilize the fact that placenta growth factor (PlGF)-1, a member of the VEGF family lacking detectable angiogenic activity, preferentially forms intracellular heterodimers with VEGF in cells coexpressing both factors. We constructed a retroviral vector containing human PlGF-1 or VEGF with a C-terminal KDEL sequence, which is a mammalian retention signal for the endoplasmic reticulum. Transduction of murine Lewis lung carcinoma cells with the retro-hPlGF-1-KDEL construct almost completely abrogated tumor growth. Consistent with the dramatic antitumor effect, most mouse VEGF molecules remained as intracellular mVEGF/hPlGF-1 heterodimers, and only a negligible amount of mVEGF homodimers were secreted. As a result, in hPlGF-1-KDEL-expressing tumors, blood vessels remained at very low numbers and lacked branching and capillary networks. Gene transfer of a hVEGF-KDEL construct into tumor cells likewise produced a dramatic antitumor effect. Thus, our study provides a novel antiangiogenic approach by preventing the secretion of VEGF.

Comparative evaluation of FGF-2-, VEGF-A-, and VEGF-C-induced angiogenesis, lymphangiogenesis, vascular fenestrations, and permeability.

Several endothelial growth factors induce both blood and lymphatic angiogenesis. However, a systematic comparative study of the impact of these factors on vascular morphology and function has been lacking. In this study, we report a quantitative analysis of the structure and macromolecular permeability of FGF-2-, VEGF-A-, and VEGF-C-induced blood and lymphatic vessels. Our results show that VEGF-A stimulated formation of disorganized, nascent vasculatures as a result of fusion of blood capillaries into premature plexuses with only a few lymphatic vessels. Ultrastructural analysis revealed that VEGF-A-induced blood vessels contained high numbers of endothelial fenestrations that mediated high permeability to ferritin, whereas the FGF-2-induced blood vessels lacked vascular fenestrations and showed only little leakage of ferritin. VEGF-C induced approximately equal amounts of blood and lymphatic capillaries with endothelial fenestrations present only on blood capillaries, mediating a medium level of ferritin leakage into the perivascular space. No endothelial fenestrations were found in FGF-2-, VEGF-A-, or VEGF-C-induced lymphatic vessels. These findings highlight the structural and functional differences between blood and lymphatic vessels induced by FGF-2, VEGF-A, and VEGF-C. Such information is important to consider in development of novel therapeutic strategies using these angiogenic factors.