<p>A functioning vasculature is critical for the supply of nutrients to other systems as well as a host of physiologic and pathologic processes. Vascular development and maintenance are tightly regulated by a number of signaling processes, among which the Tie proteins are two functioning receptors. Although they have been shown to exhibit essential roles in endothelial cell sprouting and quiescence, the mechanistic details of Tie interactions and the effects of their associations with the Angiopoietins have not been elucidated. Studies in this thesis investigated the effects of Ang2 on Tie1 activation, signaling, and cellular responses within the context of both native and immortalized endothelial cells. Additionally, we investigated the role of Ang2 in the cellular reorganization and subsequent downregulation of Tie1. We observed that Ang2, but not Ang1, induces phosphorylation of Tie1 in endothelial cells and that the extracellular domain of Tie2 is required for Ang2-mediated activation of Tie1. Furthermore, we demonstrated that Tie1 activation leads to signaling through the Akt axis, and the consequent stimulation of anti-apoptotic and pro-proliferative cellular effects. Additionally, we demonstrated that Ang2 induces a concentration and time dependent downregulation of Tie1, and that Tie2's role in the process appears to be recruitment of the ligand to the multimeric Tie complexes. Interestingly, although Ang2 stimulation is necessary, we demonstrated that Ang2 activation of Tie1 receptor complexes is not required for ligand induced downregulation of the receptor. Finally, we characterized the modulatory role of Tie1 with regards to Angiopoietin signaling through Tie2, and observed that removal of Tie1 from the surface of endothelial cells induces Ang2 activation of Tie2 leading to increases in cell survival signaling cascades. Taken together, these data shed new light on Angiopoietin signaling through the Tie receptors, further characterize the interactions between Tie1 and Tie2, suggest novel forms of endothelial cell regulation within developing and mature vasculature, and may have implications in signaling within a host of physiologic and pathologic states.</p> / Dissertation

The vascular endothelium continually senses and responds to both biochemical and mechanical stimuli to regulate vascular function in health and disease. The purpose of this dissertation was to understand the molecular mechanisms by which endothelial cells (ECs) respond to sphingosine 1-phosphate (S1P) and fluid wall shear stress (WSS) to initiate angiogenesis. To accomplish this, a novel cell culture system was developed to study the combined effects of S1P and WSS on inducing EC invasion into three-dimensional (3-D) collagen matrices. EC invasion required the presence of S1P, with the effects of S1P being enhanced by WSS to an extent comparable with S1P combined with pro-angiogenic growth factor stimulation. The extent of EC invasion depended on the magnitude of WSS in a biphasic manner, with the greatest induction occurring at 5.3 dyn/cm2 WSS. Several proteins have been implicated in EC invasion, including calpain, Akt, vimentin, p21-activated kinase (PAK), and membrane type 1-matrix metalloproteinase (MT1-MMP). Interestingly, activations of calpain and MT1-MMP and phosphorylations of Akt, PAK, and vimentin coincided with, and were required for, S1P- and WSS- induced EC invasion. Further, inhibitors of calpain, MT1-MMP, Akt and PAK all attenuated invasion induced by WSS and S1P. Calpain inhibition reduced Akt phosphorylation, vimentin cleavage, and MT1-MMP membrane translocation, suggesting that calpain regulates MT1-MMP via Akt phosphorylation and vimentin remodeling. Akt inhibition also completely blocked MT1-MMP membrane translocation and decreased phosphorylation of PAK and vimentin. In summary, these results suggest a new molecular pathway by which the combination of S1P and WSS stimulates EC invasion through calpain, Akt, PAK and vimentin to regulate activation and membrane translocation of MT1-MMP in 3-D collagen matrices.

Compelling evidence indicated that major risk factors for atherosclerosis such as oxidatively modified low density lipoprotein (oxLDL), high glucose, and reactive oxygen species promote endothelial cell apoptosis and thereby may contribute to the initiation of atherosclerotic lesion formation. Using fast protein liquid chromatography (FPLC), plasma LDL from familial hypercholesterolemic (FH) patients were separated into five subfractions, L1¡VL5. Among them, L5 subfraction was highly electronegative and suppressed DNA synthesis in cultured bovine aortic endothelial cells (BAEC) and stimulated mononuclear cell adhesion to cultured endothelial cells in vitro. Because impaired angiogenesis plays an important role in the pathogenesis of atherosclerosis, the anti-angiogenic functions of LDL subfractions from FH subjects were examined. Subconfluent BAEC (6 to 10 passages) maintained in DMEM containing 10% serum were treated with LDL subfractions at a dose of 20 £gg/ml, and the effects on anti-angiogenic functions, including cell proliferation, migration, apoptosis, tube formation, and secretion of matrix metalloproteinase (MMP) were determined. Similar to Cu2+ ox-LDL, FH-L4 and FH-L5 inhibited cell proliferation to 80.9¡Ó2.4% (p<0.05) and 58.5¡Ó4.3% of control (p<0.001), respectively, while the other FH (L1-L3) and all subfractions isolated from normocholesterolemic (N) subjects had negligible effects. Similarly, FH-L4 and -L5, but not FH-L1 to -L3, retarded cell migration to 326.9 ¡Ó 19.4 (p<0.05) and 215¡Ó16 cells (p<0.001 with the control values of 402¡Ó34 cells), respectively. FH-L5 induced almost 20% of BAEC to undergo apoptosis; FH-L4 caused very mild effects, and other subfractions did not affect apoptosis In addition, FH-L4 and -L5 perturbed tube formation by BAEC in culture (5.8¡Ó0.2 and 3.4¡Ó0.4, respectively, versus control 8.5¡Ó1.5 tubes). Finally, FH-L4 and -L5 inhibited secretion of MMP-2 by BAEC (72.7¡Ó6.9 and 18.9¡Ó4.8% of control, respectively). The results demonstrate that FH-L5 potently affects multiple processes that are vital to normal angiogenesis, FH-L4 had milder effects, and other FH and N subfractions had negligible effects. In turn, these effects in vitro on processes pivotal to angiogenesis are consistent with potential effects of ox-LDL on endothelial dysfunction during atherogenesis in vivo.

Cutaneous malignant melanoma is the fastest increasing malignancy in humans. Hepatoma-derived growth factor (HDGF) is a novel growth factor identified from human hepatoma cell line. HDGF overexpression is correlated with poor prognosis in various types of cancer including melanoma. However, the underlying mechanism of HDGF overexpression during melanoma carcinogenesis remains unclear. In this study, adding exogenous HDGF stimulated the invasion and colonies formation of B16-F10 melanoma cells. Adenovirus vectors encoding HDGF and HDGF-RNAi were generated and characterized to up- and down-regulated HDGF expression in B16-F10 melanoma cells. It was found that HDGF overexpression stimulated the proliferation, invasiveness, anchorage-independent growth of B16-F10 melanoma cells whereas HDGF knockdown exerted opposite effects. In lung-metastasis model, intravenous injection of HDGF-overexpressing melanoma cells resulted in increased metastasis while HDGF-downregulated melanoma cells caused decreased metastasis. Similarly, in primary melanoma model, subcutaneous injection of HDGF-overexpressing melanoma cells enhanced while HDGF-downregulated melanoma cells reduced the tumor burden in mice. Histological analysis revealed increased tumor proliferation and neovascularization with concomitant reduction of apoptosis in HDGF-overexpressing melanoma. Moreover, HDGF-overexpressing melanoma also exhibited enhanced propensity to metastasize from the primary tumors to lymph node and lung. Finally, it was found that HDGF overexpression increased nuclear factor kappa B (NF£eB) activities and Akt phosphorylation up and down stream alternation like PI3K, PTEN, I£eB and it¡¦s subunit IKK£\, IKK£], IKK£^ in melanoma cells. It also found that HDGF overexpression influenced MITF and HIF1£\ in melanoma after gene delivery. HDGF also altered EMT changes like E,N-cadherin, vimentin, and £],£^-catenin. The present study provides conclusive evidence that HDGF upregulation promotes the growth and metastasis of melanoma by promoting the survival and vascularization. Besides, HDGF knockdown may constitute a novel strategy for melanoma control.

High-dose morphine has been extensively used in the control of postoperative and cancer pain. Patients receiving prolonged administration of high-dose morphine are known to be associated with certain cardiovascular complications and tissue regeneration defects. This research thesis aims to investigate the biological effects and molecular mechanisms of high-dose morphine on the vascular endothelial function, angiogenesis and wound regeneration using murine models of morphine-dependence and cultured endothelial cell assays. Mice were subjected to placebo or morphine (20 mg/kg, i.p.) injection for consecutive 14 days. Aortas were harvested for assessment of vasomotor function by isometric force recordings. Protein expression p47phox (a major subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase) was determined by Western blotting. Generation of superoxide anions was detected under confocal microscope. Endothelium-dependent relaxations to acetylcholine were significantly reduced in morphine-treated animals, but were normalized by superoxide scavenging. Fluorescent densities of dihydroethidium and expression of p47phox were increased in the aorta of morphine-treated mice. In the second part of this thesis, the candidate determined the effects of high-dose morphine on angiogenesis and mobilization of endothelial progenitor cells (EPCs) in a mouse model of excisional wound injury. Excisional wound was created on control and morphine-dependent mice. Wound healing was compared by measuring the final-to-initial wound area ratio. Generation of superoxide anions in the wound was determined by luminol-enhanced chemiluminescence. Circulating mononuclear cells were isolated and measured for EPC (defined as CD34+/CD133+ cell) counts. In vivo and in vitro measurements of angiogenesis following morphine treatment were performed using the Matrigel assay. The results showed that wound closure was significantly reduced in mice treated with morphine when compared with controls, and higher levels of superoxide anions were generated in these wounds. High-dose morphine reduced numbers of circulating EPCs following creation of excisional wound. Matrigel assay showed impaired angiogenesis in animals and reduced capillary tube formation in cultured endothelial cells treated with high-concentration of morphine. Collectively, this research thesis demonstrated a number of novel findings. First, high-dose of morphine impairs vascular endothelial function by increased production of vascular superoxide anions. Activation of NADPH oxidase may be the molecular mechanisms responsible for reduced bioavailability of endothelium-derived NO. Second, systemic administration of high-dose morphine delays healing of excisional wounds and impairs angiogenesis. This antiangiogenic effect is associated with increased superoxide anions production and impaired mobilization of EPCs. In line with direct endothelial dysfunction, impaired angiogenesis and EPC mobilization resulted from high-dose morphine treatment may cause increased cardiovascular morbidity in human subjects receiving higher therapeutic dose of morphine.

Endothelial Progenitor Cells (EPCs) were first identified from human blood samples as a population of circulating mononuclear cells capable of displaying a mature endothelial cell phenotype in culture. Subsequent studies have established that EPCs arise from the bone marrow (BM) and incorporate into the endothelium at sites of blood vessel growth, suggesting a potential role for these cells in neovascularization. Furthermore, a decline in EPC count has been correlated to multiple vascular pathologies, indicating that EPC number could serve as a biomarker of cardiovascular disease. Unfortunately, due to the variability in techniques used for EPC isolation and identification, considerable heterogeneity exists within the population of cells commonly defined as EPCs. In order for the clinical potential of EPCs to be fully realized, thorough characterization of the BM-derived cell populations involved in neovascularization is required. The objective of our study was to determine the functional significance of circulating EPCs in postnatal vascular growth and repair. Two separate strategies were employed to achieve this objective. In the first, we attempted to generate a novel mouse model where the pool of bone marrow-derived endothelial precursors was drastically reduced or eliminated. Our overall approach was to deliver a "suicide" gene, under control of an endothelial cell-specific promoter, to bone marrow cells for use in bone marrow transplantation (BMT) experiments. Mice receiving BMTs would therefore lack the ability to deliver viable BM-derived EPCs to sites of neovascularization. Our central hypothesis for this study was that a reduction in EPC viability would hinder endogenous vascular repair mechanisms, thereby exacerbating cardiovascular disease. In the second strategy, we attempted to identify novel progenitor cell populations based on the transcriptional regulation of pro-angiogenic genes. Our overall approach was to transduce BM with a retrovirus containing a fluorescent reporter gene under control of pro-angiogenic promoters for use in transplantation experiments. Our central hypothesis for this study was that unique populations of BM-derived cells could be identified by expression of the fluorescent reporter gene directed by the Vascular Endothelial Growth Factor (VEGF), endothelial Nitric Oxide Synthase (eNOS) and Vascular Endothelial (VE) Cadherin promoters. The BMT strategy utilized to address our first hypothesis was unsuccessful due to the use of a truncated form of the pro-apoptotic Bax as our suicide gene target. A plasmid encoding GFP fused to the truncated Bax fragment (ΔN-Bax, consisting of amino acids 112-192 of the full length protein) was used in transfection experiments to assess ΔN-Bax function. The GFP:ΔN-Bax fusion protein formed distinct extranuclear aggregates (presumably due to mitochondrial translocation) but did not induce apoptosis in transfected cells. The ΔN-Bax fragment also did not induce cell death when targeted to endothelial cells with retoviral-mediated gene delivery or in a transgenic mouse setting. To address our second hypothesis, we generated retroviral vectors containing the fluorescent tdTomato reporter under control of the VEGF, eNOS and VE Cadherin promoters. Significant fluorescence was detected in cultured endothelial cells and ex vivo-expanded BM cells. Following transplantation of transduced BM cells into lethally irradiated recipient mice, we were able to identify circulating populations of tdTomato-positive cells using flow cytometry. With these results we have identified novel subpopulations of circulating BM-derived cells which may play a significant role in post-natal neovascularization in mice. Therefore, results acquired from these studies could lead to improved cell therapy techniques for treatment of vascular disease.

Angiogenesis by either normal or neoplastic cells involves a delicate balance of both angiogenic and angiostatic regulators. In the ovary, normal physiological angiogenesis occurs around the developing follicle and corpus luteum in response to hormonal shifts. Interestingly, carcinomas arising from the ovary are usually highly vascularized and are commonly clinically observed to produce cyst fluids or ascites which contain both angiostatic and/or angiogenic regulators. However, in contrast to normal angiogenesis, angiogenesis associated with epithelial ovarian cancer usually produces aberrant vasculature that may promote neoplastic progression. Therefore, the ovary and ovarian cancers provide models to study the mechanisms governing the strict balance of angioregulators in both normal and tumor angiogenesis. While most studies to date have focused on angiogenic regulators for normal and aberrant angiogenesis, we investigated the potential for dysregulation of angiostatic regulators to contribute to the etiology of epithelial ovarian cancer. Therefore, in this study, we examined two angiostatic regulators, angiostatin and semaphorin 3F, in epithelial ovarian cancer. Angiostatin, a cleavage product of the circulating zymogen plasminogen, was isolated from serum and urine of mice bearing a Lewis lung carcinoma and in vivo studies have demonstrated its potent angiostatic properties. Thus, we investigated the potential prognostic/diagnostic advantage of aberrant angiostatin expression with epithelial ovarian cancer. We found that urinary angiostatin, compared to other angioregulators in plasma or urine, could serve as an effective biomarker for early detection of epithelial ovarian cancer, especially when used in combination with cancer antigen 125. Additionally, urinary angiostatin correlated with both recurrent disease as well as successful tumor ablation further supporting its potential as a disease biomarker. Alternative biological functions for the axon guidance molecule, semaphorin 3F, have been reported particularly in regard to angiogenesis, tumor progression and metastasis. However, the underlying mechanisms governing semaphorin 3F regulation and dysregulation remain unclear. Therefore, we first investigated the clinical relationship between semaphorin 3F expression and epithelial ovarian cancer progression. These immunohistological studies revealed that, similar to lung cancer, semaphorin 3F expression decreased with progression supporting a tumor suppressor-like role for semaphorin 3F. Additionally, we found that calcium, an essential cellular signaling molecule, could mediate transcriptional suppression of semaphorin 3F expression in a CREB-dependent manner. Lastly, given the antagonistic relationship between semaphorin 3F and vascular endothelial growth factor, we sought to determine whether semaphorin 3F and vascular endothelial growth factor promoted opposing effects on a common downstream target. In the course of these studies we determined that telomerase is a novel molecular target of semaphorin 3F in ovarian cancer cells such that semaphorin 3F suppresses telomerase activity while vascular endothelial growth factor promotes telomerase activity. In addition, we found that the inverse relationship between semaphorin 3F and telomerase was mediated through transcriptional inhibition of the hTERT promoter by semaphorin 3F. In conclusion, this research shows that dysregulation of the angiostatic regulators, angiostatin and semaphorin 3F, may contribute to the etiology of epithelial ovarian cancer. In the future, dysregulation of these and other angiostatic regulators may be exploited for therapeutic intervention or as biomarkers for early detection which would allow women more treatment choices and hopefully, reduce the mortality associated with this insidious disease.

Multiple endocrine neoplasia syndrome type 1 (MEN1) is an autosomal dominantly inherited disease, which is described as an association of tumors mainly in endocrine organs, including pancreas and adrenal glands. Pancreatic neuroendocrine tumors (PNETs) are the most common cause of death in MEN1 patients. More than one third of the MEN1 patients also develop enlargement of the adrenals. MEN1 is caused by a germline mutation of MEN1 gene, a tumor suppressor gene that is located on the human chromosome 11. As noticed, the MEN1 related tumors often develop prior to inactivation of both wild type alleles, indicating MEN1 haploinsufficiency. In this thesis, I utilized a conventional Men1 mouse model that has the phenotype mimicking the human MEN 1 traits, in order to investigate MEN1 tumorigenesis in endocrine pancreas and adrenal glands.   The microvascular aberrations contributing to development and maintenance of PNETs were characterized. The increased vascular density of PNETs developed in the Men1 mice was paralleled by an early and extensive redistribution of pericytes within endocrine tissue. These morphological alterations were supported by fine-tuned variations in expression of several angiogenic regulators  (VEGF, FGF and PDGF) and were further potentiated by hypoxia. Vascular reactivity and blood perfusion of tumor arterioles were significantly altered in response to glucose and L-nitro-arginine methyl ester. Investigation of adrenals from10-month-old Men1 mice showed 681 proteins in mass spectrometry data sets, in which 52 proteins were commonly found in the Men1+/+ and Men1+/- adrenals, and the differential expression between the genotypes reached significant levels. Prdx3, catalyzing the reduction of oxidative stress to cell survival, is one of the overexpressed proteins. Some proteins belonging to the PPARα pathway, e.g. ACLY were also overexpressed. Subsequent microRNA (miRNA) profiling analysis of adrenals from the same age group revealed 31 miRNAs whose expression was significantly altered in comparison between the genotypes. The tumor suppressor miRNAs, miR-486, miR-330 and miR-214, were significantly downregulated in Men1+/- adrenals. The latter, miR-214, is known to inhibit ACLY expression. This finding was in concordance with the proteomic analysis. The oncogene miRNAs, miR-132 and miR-494, were significantly enhanced in the Men1+/- adrenals. Gene ontology analysis demonstrated overrepresentation of the miRNA-targeted genes that are involved in nucleic acid metabolism, vasculature development, angiogenesis, and transcription. Together, these finding after validation in humans may be exploited to improve MEN1 cancer treatment.

The cortistatins are a recently identified class of marine natural products that were found to exhibit potent and selective inhibition of human umbilical vein endothelial cells (HUVECs), making them promising leads for the development of anti-angiogenic drugs. In our synthesis, we envisioned that natural cortistatins and unnatural analogs could be prepared by late-stage introduction of isoquinolines to 17-keto precursors, and that these differentially substituted precursors could all be derived from a common key intermediate 112. We developed a robust synthetic route to prepare gram quantities of key intermediate 112 starting from readily available benzylzinc reagent 116 and enol triflate 117. Key intermediate 112 was next converted to cortistatin precursors 108, 109, 110, and 111 in three to eight steps, representing each of the four natural cortistatin ABC-ring substitution patterns. Subsequently, a generally applicable method was developed to introduce the isoquinoline moiety. After complexation to N,N,N',N'-tetramethylethylenediamine (TMEDA), 7-lithio-isoquinoline added to 17-keto precursors to provide the corresponding 1,2-addition products; the resulting tertiary alcohols underwent radical deoxygenation via their trifluoroacetates to afford the desired (17S)-products. This organolithium-addition-deoxygenation sequence provided cortistatins A (1, on a 20-mg scale), J (9), K (10), and L (11) in good overall yields. We also synthesized cortistatin primary amines (176 and 186) and used them to prepare several cortistatin based affinity reagents. By employing these reagents in pull-down experiments, we identified a 55-kD membrane kinase as a putative protein target of cortistatins. We wanted to prepare cortistatin analogs with isoquinoline modifications due to the importance of this ring for the biological activity of cortistatins. This led us to develop a novel and versatile synthesis of substituted isoquinolines. In our method, lithiated o-tolualdehyde tert-butylimines were condensed with different nitriles to generate eneamido anion intermediates, which were trapped in situ with various electrophiles at the C4-position, affording a wide range of substituted isoquinolines. Further diversification was achieved by modification of the work-up conditions and by subsequent transformations. / Chemistry and Chemical Biology

Η περιφερική αρτηριοπάθεια αποτελεί μία νόσο με αυξημένη επίπτωση στις σύγχρονες χώρες. Οι τρόποι αντιμετώπισής της ποικίλουν, ωστόσο, ένα μεγάλο ποσοστό ασθενών δεν μπορεί να αντιμετωπιστεί με τις υπάρχουσες συμβατικές θεραπείες, είτε χειρουργικές, είτε συντηρητικές. Η αγγειογένεση αποτελεί έναν πολλά υποσχόμενο τρόπο αντιμετώπισης. Επιτελείται ευρεία έρευνα με χρησιμοποίηση μιας μεγάλης ποικιλίας ουσιών και κυττάρων με στόχο την επίτευξη θεραπευτικής αγγειογένεσης που θα βελτιώσει την κλινική εικόνα των ασθενών με περιφερική αρτηριοπάθεια. Ο νευρικός αυξητικός παράγοντας (NGF) υπάρχουν αναφορές ότι έχει σημαντικό ρόλο στη φυσιολογική και παθολογική αγγειογένεση. Βασισμένοι σε αυτές τις παρατηρήσεις, υποθέσαμε ότι ο NGF μπορεί να επάγει το σχηματισμό λειτουργικών αιμοφόρων αγγείων σε μοντέλο ισχαιμίας οπισθίων άκρων κονίκλου. Η ισχαιμία στα οπίσθια άκρα προκλήθηκε σε 34 κονίκλους με αμφοτερόπλευρο εμβολισμό της μηριαίας αρτηρίας. Την 7η, 14η και 20η ημέρα μετά τον εμβολισμό χορηγήθηκε NGF ενδομυϊκά σε ένα από τα δύο ισχαιμικά άκρα και εγχύθηκε μάρτυρας στο άλλο άκρο. Την 40η ημέρα, τα νεοσχηματισμένα παράπλευρα αγγεία διαμέτρου μεγαλύτερης των 500μm ποσοτικοποιήθηκαν με διαωτιαία ενδοαρτηριακή αφαιρετική αγγειογραφία. Πραγματοποιήθηκε, επίσης, in vivo δυναμική αξονική τομογραφία αιμάτωσης σε αμφότερα τα άκρα ώστε να διερευνηθεί η αιμοδυναμική ανάρρωση των ισχαιμικών ιστών. Η λειτουργική εκτίμηση της αιμάτωσης των άκρων έδειξε στατιστικά σημαντική αύξηση της αιματικής ροής και του όγκου αίματος στα άκρα που χορηγήθηκε NGF. Ωστόσο, η αύξηση των παράπλευρων αγγείων δεν ήταν ανιχνεύσιμη αγγειογραφικά, κάτι που υποδηλώνει ότι ο NGF ενίσχυσε τη δημιουργία τριχοειδικού δικτύου αλλά όχι την αρτηριογένεση. Ο συνδυασμός του NGF είτε με αναστολέα του TrkA είτε με αναστολέα του VEGFR-2, κατήργησε την αιμοδυναμική ανάρρωση που προκάλεσε ο NGF. Αυτό δείχνει εξάρτηση της αγγειογενετικής δράσης του NGF τόσο από το δικό του υποδοχέα άμεσα, όσο και από τον υποδοχέα του VEGF έμμεσα. Τα ανωτέρω ευρήματα προσφέρουν νέους ορίζοντες στην κατανόηση της δράσης του NGF στο σχηματισμό νέων αγγείων και στις πιθανές εφαρμογές του στη θεραπευτική αγγειογένεση. / Nerve growth factor (NGF) has been reported to play an important role in physiological and pathological angiogenesis. Based on these observations, we hypothesized that NGF may induce the formation of functional blood vessels in a hindlimb ischemic rabbit model. Hindlimb ischemia was induced in 34 rabbits bilaterally by endovascular embolization of femoral arteries. On the 7th, 14th, and 20th postembolization days, NGF was injected intramuscularly, in 1 ischemic limb, and vehicle was injected in the contralateral control limb. On the 40th day, newly developed collateral vessels (diameter .500 mm) were quantified by transauricular intraarterial subtraction angiography. Perfusion analysis of an in vivo dynamic computed tomography study was performed to the limbs to investigate the hemodynamic recovery of the distal ischemic tissues. Functional estimation of limb perfusion showed a statistically significant increase of blood flow and blood volume for NGF. However, the increase of the collateral vessels was not detectable angiographically, providing evidence for the existence of a NGF-stimulated capillary angiogenic network but not increase of arteriogenesis. The combination of NGF with either tropomyosin-related kinase type A or vascular endothelial growth factor receptor 2 antagonists abolished the NGF-induced hemodynamic recovery. These findings provide new insights into understanding the involvement of NGF in vascular formation and its applications in therapeutic angiogenesis.