Roberto Nicosia - Academia.edu (original) (raw)

Papers by Roberto Nicosia

Supplementary Figure 1 from Transforming Growth Factor-β1 and CD105 Promote the Migration of Hepa... more Supplementary Figure 1 from Transforming Growth Factor-β1 and CD105 Promote the Migration of Hepatocellular Carcinoma–Derived Endothelium

Arteriosclerosis, Thrombosis, and Vascular Biology, 2015

Introduction: Atherosclerotic plaque rupture is the most common trigger of myocardial infarctions... more Introduction: Atherosclerotic plaque rupture is the most common trigger of myocardial infarctions and strokes. The molecular mechanisms of plaque rupture are uncertain; accordingly, there are no specific interventions aimed at preventing plaque rupture. We used unbiased “shotgun” proteomics to identify proteins and pathways that are associated with human plaque rupture. We performed parallel proteomic analyses in a mouse model that develops plaque rupture. Methods: We used tandem mass spectrometry, performed on tissue extracts, to compare the proteomes of: 1) stable and unstable areas of human carotid plaques (n = 6); and 2) aortic arches of ApoE-null mice with (n = 6) or without (n = 6) macrophage-specific overexpression of urokinase plasminogen activator (uPA). Protein abundance was compared using the PepC statistical program, with correction for multiple comparisons. Gene ontology and pathway enrichment analysis were performed with DAVID. Results: Mass spectrometry detected a tot...

Angiogenesis, 2016

This study was designed to investigate how changes in O2 levels affected angiogenesis in vascular... more This study was designed to investigate how changes in O2 levels affected angiogenesis in vascular organ culture. Although hypoxia is a potent inducer of angiogenesis, aortic rings cultured in collagen paradoxically failed to produce an angiogenic response in 1-4 % O2. Additionally, aortic neovessels preformed in atmospheric O2 lost pericytes and regressed at a faster rate than control when exposed to hypoxia. Aortic explants remained viable in hypoxia and produced an angiogenic response when returned to atmospheric O2. Hypoxic aortic rings were unresponsive to VEGF, while increased oxygenation of the system dose-dependently enhanced VEGF-induced angiogenesis. Hypoxia-induced refractoriness to angiogenic stimulation was not restricted to the aorta because similar results were obtained with vena cava explants or isolated endothelial cells. Unlike endothelial cells, aorta-derived mural cells were unaffected by hypoxia. Hypoxia downregulated expression in aortic explants of key signalin...

Methods in molecular biology (Clifton, N.J.), 2015

Protocols outlined in this chapter illustrate how to prepare and analyze angiogenic cultures of r... more Protocols outlined in this chapter illustrate how to prepare and analyze angiogenic cultures of rat aorta. Aortic rings embedded in gels of extracellular matrix generate vascular outgrowths that can easily be monitored over time with inverted microscopy. The angiogenic response can be measured by counting vessels or with image analysis. Aortic ring cultures can be used to investigate molecular mechanisms of angiogenesis and test the efficacy of stimulators and inhibitors of the angiogenic process. As such this assay is an invaluable tool for both basic and applied angiogenesis research.

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 5847 Hepatocellular carcinoma (HCC) is one o... more AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 5847 Hepatocellular carcinoma (HCC) is one of most malignant and aggressive human cancers. Transforming growth factor beta 1 (TGF-β1) is believed to contribute to the progression of this neoplasm. CD105 (Endoglin), a component of the TGF-β1 receptor complex, is expressed on endothelial cells (EC) during angiogenesis particularly in tumors. To investigate the role of TGF-β1 and CD105 in HCC-related angiogenesis we isolated and cultured EC from HCC and adjacent non-neoplastic liver (nNL). Compared to nNL-derived EC (nNL-EC), EC derived from HCC (HCC-EC) overexpressed CD105. Recombinant TGF-β1 did not affect HCC-EC proliferation but slightly augmented the effect of VEGF-A. Compared to nNL-EC, HCC-EC had a higher spontaneous motility which was enhanced by a fibronectin (FN) matrix. The addition of high dose TGF-β1 as chemotaxic factor (20-50ng/ml) markedly increased migration of HCC-EC, but had little or no effect on nNL-EC. The chemotactic effect of TGF-β1 correlated with the expression of CD105 in HCC-EC and the grade of HCC malignancy. Monoclonal antibodies against CD105 blocked spontaneous and TGF-β1-induced migration. Histological examination of 24 different HCC biopsies demonstrated that the expression of CD105 and TGF-β1 correlated with higher expression of vascular markers such as CD44 and VE-cad, increased microvascular density (MVD), and the grade of tumor malignancy. In high grade HCC, CD105 was preferentially expressed in the “activated” vessels at the tumor periphery, while TGF-β1 was highly expressed in HCC and absent in the adjacent nNL. The most malignant HCC showed cytoplasmic and nuclear expression of β-catenin (β-cat) and down modulation of E-cadherins (E-cad), consistent with an invasive phenotype. In summary our studies indicate that HCC cells overexpress TGF-β1 while HCC-EC overexpress CD105 which is necessary for spontaneous and TGF-β1-stimulated HCC-EC migration. These findings suggest that the TGF-β1/CD105 system plays an important role in HCC-related angiogenesis and tumor progression.

Angiogenesis, 2003

Angiogenesis can be studied ex vivo by culturing rat or mouse aortic rings in collagen gels. Unli... more Angiogenesis can be studied ex vivo by culturing rat or mouse aortic rings in collagen gels. Unlike rat aorta explants, unstimulated mouse aortic rings were unable to spontaneously produce an angiogenic response under serum-free conditions. They, however, responded to bFGF and VEGF, generating networks of branching neovessels. Aortic rings from GFP-Tie2-transgenic mice generated GFP-labeled neovessels that could be easily identified by their distinctly green fluorescence. Aortic rings from 1-to 2-month-old mice produced microvessels faster, more uniformly and in greater number than aortic rings from 6-to 10-month-old mice, particularly in VEGF-treated cultures. Aortic rings from 129/SVJ mice were capable of a much stronger and sustained angiogenic response to bFGF than those of C57BL/6 or BALB/c mice, which were in turn more angiogenic than aortic rings from FVB mice. The same strains of mice responded differently to VEGF, as C57BL/6 mouse aortic rings produced more microvessels than those of BALB/c, FVB, and 129/SVJ mice, which were capable of only a limited response. The significant impact that aging and genetic background have on mouse aortic angiogenesis should be taken into account when the aortic-ring assay is used to evaluate function of genes that have been deleted or overexpressed in genetically modified mice.

Hepatocellular carcinoma (HCC) is one of most malignant and aggressive human tumors. Transforming... more Hepatocellular carcinoma (HCC) is one of most malignant and aggressive human tumors. Transforming growth factor-B1 (TGF-B1) and its coreceptor CD105have been shown to contribute to HCC malignant progression. TGF-B1 and CD105 have also been implicated in angiogenesis, but their role in the vascularization of HCC has not been investigated. To fill this gap, we studied the effect of TGF-B1 and CD105on HCC-derived endothelium. By using immunomagnetic beads, we isolated and cultured endothelial cells (ECs) from HCC (HCC-EC) and adjacent nonneoplastic tissue (nNL-ECs) obtained from 24 liver biopsies. HCC and nNL biopsies were also analyzed by immunohistochemistry for the expression of CD105, TGF-B1, Ve-cadherin (Ve-cad), CD44, B-catenin, and E-cadherin. Com- pared with nNL-ECs, HCC-ECs had higher expression of CD105, enhanced spontaneous motility, and greater capacity to migrate in response to TGF-B1 (5ng/mL), particularly in the presence of a fibronectin matrix. The chemotactic effect of...

The Journal of Cell Biology, 1998

The αvβ3 integrin plays a fundamental role during the angiogenesis process by inhibiting endothel... more The αvβ3 integrin plays a fundamental role during the angiogenesis process by inhibiting endothelial cell apoptosis. However, the mechanism of inhibition is unknown. In this report, we show that integrin-mediated cell survival involves regulation of nuclear factor-kappa B (NF-κB) activity. Different extracellular matrix molecules were able to protect rat aorta- derived endothelial cells from apoptosis induced by serum withdrawal. Osteopontin and β3 integrin ligation rapidly increased NF-κB activity as measured by gel shift and reporter activity. The p65 and p50 subunits were present in the shifted complex. In contrast, collagen type I (a β1-integrin ligand) did not induce NF-κB activity. The αvβ3 integrin was most important for osteopontin-mediated NF-κB induction and survival, since adding a neutralizing anti-β3 integrin antibody blocked NF-κB activity and induced endothelial cell death when cells were plated on osteopontin. NF-κB was required for osteopontin- and vitronectin-induc...

Laboratory Investigation, 2001

Vasculogenesis, the de novo formation of new blood vessels from undifferentiated precursor cells ... more Vasculogenesis, the de novo formation of new blood vessels from undifferentiated precursor cells or angioblasts, has been studied with experimental in vivo and ex vivo animal models, but its mechanism is poorly understood, particularly in humans. We used the aortic ring assay to investigate the angioforming capacity of aortic explants from 11-to 12-week-old human embryos. After being embedded in collagen gels, the aorta rings produced branching capillary-like structures formed by mesenchymal spindle cells that lined a capillary-like lumen and expressed markers of endothelial differentiation (CD31, CD34, von Willebrand factor [vWF], and fms-like tyrosine kinase-1 [Flk-1]/vascular endothelial growth factor receptor 2 [VEGFR2]). The cell linings of these structures showed ultrastructural evidence of endothelial differentiation. The neovascular proliferation occurred primarily in the outer aspects of aortic rings, thus suggesting that the new vessels mainly arose from immature endothelial precursor cells localized in the outer layer of the aortic stroma, ie, a process of vasculogenesis rather than angiogenesis. The undifferentiated mesenchymal cells (CD34ϩ/CD31Ϫ), isolated and cultured on collagen-fibronectin, differentiated into endothelial cells expressing CD31 and vWF. Furthermore, the CD34ϩ/CD31ϩ cells were capable of forming a network of capillary-like structures when cultured on Matrigel. This is the first reported study showing the ex vivo formation of human microvessels by vasculogenesis. Our findings indicate that the human embryonic aorta is a rich source of CD34ϩ/CD31Ϫ endothelial progenitor cells (angioblasts), and this information may prove valuable in studies of vascular regeneration and tissue bioengineering. (Lab Invest 2001, 81:875-885).

Journal of Cellular and Molecular Medicine, 2009

Introduction Summary and conclusion The aortic ring model has become one of the most widely used ... more Introduction Summary and conclusion The aortic ring model has become one of the most widely used methods to study angiogenesis and its mechanisms. Many factors have contributed to its popularity including reproducibility, cost effectiveness, ease of use and good correlation with in vivo studies. In this system aortic rings embedded in biomatrix gels and cultured under chemically defined conditions generate arborizing vascular outgrowths which can be stimulated or inhibited with angiogenic regulators. Originally based on the rat aorta, the aortic ring model was later adapted to the mouse for the evaluation of specific molecular alterations in genetically modified animals. Viral transduction of the aortic rings has enabled investigators to overexpress genes of interest in the aortic cultures. Experiments on angiogenic mechanisms have demonstrated that formation of neovessels in aortic cultures is regulated by macrophages, pericytes and fibroblasts through a complex molecular cascade i...

In Vitro Cellular & Developmental Biology - Animal, 2000

Type IV collagen is a major basement membrane component that has been implicated in the regulatio... more Type IV collagen is a major basement membrane component that has been implicated in the regulation of angiogenesis. The purpose of this study was to evaluate the effect of type IV collagen on the angiogenic response of native endothelial cells in three-dimensional vascular organ culture. Rings of rat aorta were cultured under serum-free conditions in gels of type I collagen with or without type IV collagen. In the absence of type IV collagen, aortic rings generated neovessels, which proliferated until day 9 and gradually regressed during the second and third weeks of culture. Type IV collagen promoted neovessel elongation and survival in a dose-dependent manner. Microvascular length increased by 43, 57, and 119% over control values in cultures treated with 3, 30, and 300 p,g/ml type IV collagen, respectively. When used at high concentrations (300 ~g/ml) type IV collagen stabilized the ne0vascular outgrowths and prevented vascular regression. Type IV collagen also promoted the formation of neovessels, but significant stimulatory effects were observed only at an intermediate concentration (30 p~g/ml) and were no longer significant at the high concentration (300 txg/ml). The observation that type IV collagen has dose-dependent effects on vascular elongation, proliferation, and stabilization, supports the concept that the developing basement membrane of neovessels acts as a solid-phase regulator of angiogenesis, whose function varies depending on the concentration of its molecular components.

Gynecologic Oncology, 2010

The poor prognosis associated with epithelial ovarian cancer (EOC) is due to the lack of overt ea... more The poor prognosis associated with epithelial ovarian cancer (EOC) is due to the lack of overt early symptoms and the absence of reliable diagnostic screening methods. Since many tumors over express angiogenic regulators, the purpose of this study was to determine whether elevated levels of the angiogenic or angiostatic molecules vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), endostatin (ES), and angiostatin (AS) were elevated in plasma and urine from patients with EOC. VEGF, HGF, ES and AS were assayed by ELISA in samples from pilot cohort consisting of healthy women (N=48; pre-menopausal N=23, post-menopausal N=25), women with benign gynecological disease (N=54), patients with primary peritoneal cancer (PP) (N=2) and EOC (N=35). Wherever possible, parallel serum samples were measured for CA125 levels by ELISA. AS was the angioregulator that independently discriminated EOC patients from healthy individuals. Levels of urinary AS (uAS) from healthy individuals or women with benign gynecological disease averaged 21.4 ng/mL+/-3.7 and 41.5 ng/mL+/-8.8, respectively. In contrast, uAS averaged 115 ng/mL+/-39.2 and 276 ng/mL+/-45.8 from women with Stage I (N=6) and late stage (N=31) EOC, respectively. Furthermore, uAS was elevated in EOC patients regardless of tumor grade, stage, size, histological subtype, creatinine levels, menopausal status, or patient age, but appeared to complement CA125 measurements. Levels of AS are elevated in the urine of patients with EOC and may be of diagnostic and/or prognostic clinical importance. Further studies of uAS as a biomarker for EOC alone or in combination with other markers are warranted.

Current Pharmaceutical Design, 2009

Cancer Research, 2008

Hepatocellular carcinoma (HCC) is one of most malignant and aggressive human tumors. Transforming... more Hepatocellular carcinoma (HCC) is one of most malignant and aggressive human tumors. Transforming growth factor-β1 (TGF-β1) and its coreceptor CD105 have been shown to contribute to HCC malignant progression. TGF-β1 and CD105 have also been implicated in angiogenesis, but their role in the vascularization of HCC has not been investigated. To fill this gap, we studied the effect of TGF-β1 and CD105 on HCC-derived endothelium. By using immunomagnetic beads, we isolated and cultured endothelial cells (ECs) from HCC (HCC-EC) and adjacent nonneoplastic tissue (nNL-ECs) obtained from 24 liver biopsies. HCC and nNL biopsies were also analyzed by immunohistochemistry for the expression of CD105, TGF-β1, Ve-cadherin (Ve-cad), CD44, β-catenin, and E-cadherin. Compared with nNL-ECs, HCC-ECs had higher expression of CD105, enhanced spontaneous motility, and greater capacity to migrate in response to TGF-β1 (5 ng/mL), particularly in the presence of a fibronectin matrix. The chemotactic effect o...

Arteriosclerosis, Thrombosis, and Vascular Biology, 2011

Objective—The goal of this study was to define the role of tumor necrosis factor-α (TNFα) in the ... more Objective—The goal of this study was to define the role of tumor necrosis factor-α (TNFα) in the cascade of gene activation that regulates aortic angiogenesis in response to injury.Methods and Results—Angiogenesis was studied by culturing rat or mouse aortic rings in collagen gels. Gene expression was evaluated by quantitative reverse transcription–polymerase chain reaction, microarray analysis, immunocytochemistry, and ELISA. TNFα gene disruption and recombinant TNFα or blocking antibodies against vascular endothelial growth factor (VEGF) or TNF receptors were used to investigate TNFα-mediated angiogenic mechanisms. Resident aortic macrophages were depleted with liposomal clodronate. Angiogenesis was preceded by overexpression of TNFα and TNFα-inducible genes. Studies with isolated cells showed that macrophages were the main source of TNFα. Angiogenesis, VEGF production, and macrophage outgrowth were impaired by TNFα gene disruption and promoted by exogenous TNFα. Antibody-mediated...

Angiogenesis, 2007

Rat or mouse aortic rings produce angiogenic outgrowths in vitro through endogenous production of... more Rat or mouse aortic rings produce angiogenic outgrowths in vitro through endogenous production of growth factors and inflammatory cytokines. To further investigate this process in vivo, collagen-Gelfoam constructs containing aortic rings were implanted subcutaneously in syngeneic animals. Aortic rings stimulated a prominent angiogenic response characterized by peri- and intra-aortic accumulation of florid granulation tissue. Conversely, implants without rings elicited a non-specific inflammatory reaction without significant angiogenesis. The angiogenic response to the rings peaked at day 14 and was followed by regression of neovessels, which were mostly reabsorbed by day 28. Gene expression studies showed upregulated expression of angiogenic growth factors and cytokines in implants with rings. Tracking experiments with LacZ expressing ROSA26 transgenic mice demonstrated that both the aorta and the host contributed to the angiogenic response. These studies show that the angiogenic properties of the rodent aorta can be studied in the live animal under conditions that can be monitored and quantified. This in vivo assay can be used to study the molecular mechanisms by which the arterial wall and its proangiogenic cytokines regulate formation of granulation tissue during wound healing.

The American Journal of Pathology, 2007

Vasculogenesis, the formation of blood vessels in embryonic or fetal tissue mediated by immature ... more Vasculogenesis, the formation of blood vessels in embryonic or fetal tissue mediated by immature vascular cells (ie, angioblasts), is poorly understood. We report the identification of a population of vascular progenitor cells (hVPCs) in the human fetal aorta composed of undifferentiated mesenchymal cells that coexpress endothelial and myogenic markers. Under culture conditions that promoted cell differentiation, hVPCs gave rise to a mixed population of mature endothelial and mural cells when progenitor cells were stimulated with vascular endothelial growth factor-A or platelet-derived growth factor-␤␤. hVPCs grew as nonadherent cells and, when embedded in a three-dimensional collagen gel, reorganized into cohesive cellular cords that resembled mature vascular structures. hVPC-conditioned medium contained angiogenic substances (vascular endothelial growth factor-A and angiopoietin-2) and strongly stimulated the proliferation of endothelial cells. We also demonstrate the therapeutic efficacy of a small number of hVPCs transplanted into ischemic limb muscle of immunodeficient mice. hVPCs markedly improved neovascularization and inhibited the loss of endogenous endothelial cells and myocytes, thus ameliorating the clinical outcome from ischemia. We conclude that fetal aorta represents an important source for the investigation of the phenotypic and functional features of human vascular progenitor cells.

American Journal of Kidney Diseases, 2006

Supplementary Figure 1 from Transforming Growth Factor-β1 and CD105 Promote the Migration of Hepa... more Supplementary Figure 1 from Transforming Growth Factor-β1 and CD105 Promote the Migration of Hepatocellular Carcinoma–Derived Endothelium

Arteriosclerosis, Thrombosis, and Vascular Biology, 2015

Introduction: Atherosclerotic plaque rupture is the most common trigger of myocardial infarctions... more Introduction: Atherosclerotic plaque rupture is the most common trigger of myocardial infarctions and strokes. The molecular mechanisms of plaque rupture are uncertain; accordingly, there are no specific interventions aimed at preventing plaque rupture. We used unbiased “shotgun” proteomics to identify proteins and pathways that are associated with human plaque rupture. We performed parallel proteomic analyses in a mouse model that develops plaque rupture. Methods: We used tandem mass spectrometry, performed on tissue extracts, to compare the proteomes of: 1) stable and unstable areas of human carotid plaques (n = 6); and 2) aortic arches of ApoE-null mice with (n = 6) or without (n = 6) macrophage-specific overexpression of urokinase plasminogen activator (uPA). Protein abundance was compared using the PepC statistical program, with correction for multiple comparisons. Gene ontology and pathway enrichment analysis were performed with DAVID. Results: Mass spectrometry detected a tot...

Angiogenesis, 2016

This study was designed to investigate how changes in O2 levels affected angiogenesis in vascular... more This study was designed to investigate how changes in O2 levels affected angiogenesis in vascular organ culture. Although hypoxia is a potent inducer of angiogenesis, aortic rings cultured in collagen paradoxically failed to produce an angiogenic response in 1-4 % O2. Additionally, aortic neovessels preformed in atmospheric O2 lost pericytes and regressed at a faster rate than control when exposed to hypoxia. Aortic explants remained viable in hypoxia and produced an angiogenic response when returned to atmospheric O2. Hypoxic aortic rings were unresponsive to VEGF, while increased oxygenation of the system dose-dependently enhanced VEGF-induced angiogenesis. Hypoxia-induced refractoriness to angiogenic stimulation was not restricted to the aorta because similar results were obtained with vena cava explants or isolated endothelial cells. Unlike endothelial cells, aorta-derived mural cells were unaffected by hypoxia. Hypoxia downregulated expression in aortic explants of key signalin...

Methods in molecular biology (Clifton, N.J.), 2015

Protocols outlined in this chapter illustrate how to prepare and analyze angiogenic cultures of r... more Protocols outlined in this chapter illustrate how to prepare and analyze angiogenic cultures of rat aorta. Aortic rings embedded in gels of extracellular matrix generate vascular outgrowths that can easily be monitored over time with inverted microscopy. The angiogenic response can be measured by counting vessels or with image analysis. Aortic ring cultures can be used to investigate molecular mechanisms of angiogenesis and test the efficacy of stimulators and inhibitors of the angiogenic process. As such this assay is an invaluable tool for both basic and applied angiogenesis research.

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 5847 Hepatocellular carcinoma (HCC) is one o... more AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 5847 Hepatocellular carcinoma (HCC) is one of most malignant and aggressive human cancers. Transforming growth factor beta 1 (TGF-β1) is believed to contribute to the progression of this neoplasm. CD105 (Endoglin), a component of the TGF-β1 receptor complex, is expressed on endothelial cells (EC) during angiogenesis particularly in tumors. To investigate the role of TGF-β1 and CD105 in HCC-related angiogenesis we isolated and cultured EC from HCC and adjacent non-neoplastic liver (nNL). Compared to nNL-derived EC (nNL-EC), EC derived from HCC (HCC-EC) overexpressed CD105. Recombinant TGF-β1 did not affect HCC-EC proliferation but slightly augmented the effect of VEGF-A. Compared to nNL-EC, HCC-EC had a higher spontaneous motility which was enhanced by a fibronectin (FN) matrix. The addition of high dose TGF-β1 as chemotaxic factor (20-50ng/ml) markedly increased migration of HCC-EC, but had little or no effect on nNL-EC. The chemotactic effect of TGF-β1 correlated with the expression of CD105 in HCC-EC and the grade of HCC malignancy. Monoclonal antibodies against CD105 blocked spontaneous and TGF-β1-induced migration. Histological examination of 24 different HCC biopsies demonstrated that the expression of CD105 and TGF-β1 correlated with higher expression of vascular markers such as CD44 and VE-cad, increased microvascular density (MVD), and the grade of tumor malignancy. In high grade HCC, CD105 was preferentially expressed in the “activated” vessels at the tumor periphery, while TGF-β1 was highly expressed in HCC and absent in the adjacent nNL. The most malignant HCC showed cytoplasmic and nuclear expression of β-catenin (β-cat) and down modulation of E-cadherins (E-cad), consistent with an invasive phenotype. In summary our studies indicate that HCC cells overexpress TGF-β1 while HCC-EC overexpress CD105 which is necessary for spontaneous and TGF-β1-stimulated HCC-EC migration. These findings suggest that the TGF-β1/CD105 system plays an important role in HCC-related angiogenesis and tumor progression.

Angiogenesis, 2003

Angiogenesis can be studied ex vivo by culturing rat or mouse aortic rings in collagen gels. Unli... more Angiogenesis can be studied ex vivo by culturing rat or mouse aortic rings in collagen gels. Unlike rat aorta explants, unstimulated mouse aortic rings were unable to spontaneously produce an angiogenic response under serum-free conditions. They, however, responded to bFGF and VEGF, generating networks of branching neovessels. Aortic rings from GFP-Tie2-transgenic mice generated GFP-labeled neovessels that could be easily identified by their distinctly green fluorescence. Aortic rings from 1-to 2-month-old mice produced microvessels faster, more uniformly and in greater number than aortic rings from 6-to 10-month-old mice, particularly in VEGF-treated cultures. Aortic rings from 129/SVJ mice were capable of a much stronger and sustained angiogenic response to bFGF than those of C57BL/6 or BALB/c mice, which were in turn more angiogenic than aortic rings from FVB mice. The same strains of mice responded differently to VEGF, as C57BL/6 mouse aortic rings produced more microvessels than those of BALB/c, FVB, and 129/SVJ mice, which were capable of only a limited response. The significant impact that aging and genetic background have on mouse aortic angiogenesis should be taken into account when the aortic-ring assay is used to evaluate function of genes that have been deleted or overexpressed in genetically modified mice.

Hepatocellular carcinoma (HCC) is one of most malignant and aggressive human tumors. Transforming... more Hepatocellular carcinoma (HCC) is one of most malignant and aggressive human tumors. Transforming growth factor-B1 (TGF-B1) and its coreceptor CD105have been shown to contribute to HCC malignant progression. TGF-B1 and CD105 have also been implicated in angiogenesis, but their role in the vascularization of HCC has not been investigated. To fill this gap, we studied the effect of TGF-B1 and CD105on HCC-derived endothelium. By using immunomagnetic beads, we isolated and cultured endothelial cells (ECs) from HCC (HCC-EC) and adjacent nonneoplastic tissue (nNL-ECs) obtained from 24 liver biopsies. HCC and nNL biopsies were also analyzed by immunohistochemistry for the expression of CD105, TGF-B1, Ve-cadherin (Ve-cad), CD44, B-catenin, and E-cadherin. Com- pared with nNL-ECs, HCC-ECs had higher expression of CD105, enhanced spontaneous motility, and greater capacity to migrate in response to TGF-B1 (5ng/mL), particularly in the presence of a fibronectin matrix. The chemotactic effect of...

The Journal of Cell Biology, 1998

The αvβ3 integrin plays a fundamental role during the angiogenesis process by inhibiting endothel... more The αvβ3 integrin plays a fundamental role during the angiogenesis process by inhibiting endothelial cell apoptosis. However, the mechanism of inhibition is unknown. In this report, we show that integrin-mediated cell survival involves regulation of nuclear factor-kappa B (NF-κB) activity. Different extracellular matrix molecules were able to protect rat aorta- derived endothelial cells from apoptosis induced by serum withdrawal. Osteopontin and β3 integrin ligation rapidly increased NF-κB activity as measured by gel shift and reporter activity. The p65 and p50 subunits were present in the shifted complex. In contrast, collagen type I (a β1-integrin ligand) did not induce NF-κB activity. The αvβ3 integrin was most important for osteopontin-mediated NF-κB induction and survival, since adding a neutralizing anti-β3 integrin antibody blocked NF-κB activity and induced endothelial cell death when cells were plated on osteopontin. NF-κB was required for osteopontin- and vitronectin-induc...

Laboratory Investigation, 2001

Vasculogenesis, the de novo formation of new blood vessels from undifferentiated precursor cells ... more Vasculogenesis, the de novo formation of new blood vessels from undifferentiated precursor cells or angioblasts, has been studied with experimental in vivo and ex vivo animal models, but its mechanism is poorly understood, particularly in humans. We used the aortic ring assay to investigate the angioforming capacity of aortic explants from 11-to 12-week-old human embryos. After being embedded in collagen gels, the aorta rings produced branching capillary-like structures formed by mesenchymal spindle cells that lined a capillary-like lumen and expressed markers of endothelial differentiation (CD31, CD34, von Willebrand factor [vWF], and fms-like tyrosine kinase-1 [Flk-1]/vascular endothelial growth factor receptor 2 [VEGFR2]). The cell linings of these structures showed ultrastructural evidence of endothelial differentiation. The neovascular proliferation occurred primarily in the outer aspects of aortic rings, thus suggesting that the new vessels mainly arose from immature endothelial precursor cells localized in the outer layer of the aortic stroma, ie, a process of vasculogenesis rather than angiogenesis. The undifferentiated mesenchymal cells (CD34ϩ/CD31Ϫ), isolated and cultured on collagen-fibronectin, differentiated into endothelial cells expressing CD31 and vWF. Furthermore, the CD34ϩ/CD31ϩ cells were capable of forming a network of capillary-like structures when cultured on Matrigel. This is the first reported study showing the ex vivo formation of human microvessels by vasculogenesis. Our findings indicate that the human embryonic aorta is a rich source of CD34ϩ/CD31Ϫ endothelial progenitor cells (angioblasts), and this information may prove valuable in studies of vascular regeneration and tissue bioengineering. (Lab Invest 2001, 81:875-885).

Journal of Cellular and Molecular Medicine, 2009

Introduction Summary and conclusion The aortic ring model has become one of the most widely used ... more Introduction Summary and conclusion The aortic ring model has become one of the most widely used methods to study angiogenesis and its mechanisms. Many factors have contributed to its popularity including reproducibility, cost effectiveness, ease of use and good correlation with in vivo studies. In this system aortic rings embedded in biomatrix gels and cultured under chemically defined conditions generate arborizing vascular outgrowths which can be stimulated or inhibited with angiogenic regulators. Originally based on the rat aorta, the aortic ring model was later adapted to the mouse for the evaluation of specific molecular alterations in genetically modified animals. Viral transduction of the aortic rings has enabled investigators to overexpress genes of interest in the aortic cultures. Experiments on angiogenic mechanisms have demonstrated that formation of neovessels in aortic cultures is regulated by macrophages, pericytes and fibroblasts through a complex molecular cascade i...

In Vitro Cellular & Developmental Biology - Animal, 2000

Type IV collagen is a major basement membrane component that has been implicated in the regulatio... more Type IV collagen is a major basement membrane component that has been implicated in the regulation of angiogenesis. The purpose of this study was to evaluate the effect of type IV collagen on the angiogenic response of native endothelial cells in three-dimensional vascular organ culture. Rings of rat aorta were cultured under serum-free conditions in gels of type I collagen with or without type IV collagen. In the absence of type IV collagen, aortic rings generated neovessels, which proliferated until day 9 and gradually regressed during the second and third weeks of culture. Type IV collagen promoted neovessel elongation and survival in a dose-dependent manner. Microvascular length increased by 43, 57, and 119% over control values in cultures treated with 3, 30, and 300 p,g/ml type IV collagen, respectively. When used at high concentrations (300 ~g/ml) type IV collagen stabilized the ne0vascular outgrowths and prevented vascular regression. Type IV collagen also promoted the formation of neovessels, but significant stimulatory effects were observed only at an intermediate concentration (30 p~g/ml) and were no longer significant at the high concentration (300 txg/ml). The observation that type IV collagen has dose-dependent effects on vascular elongation, proliferation, and stabilization, supports the concept that the developing basement membrane of neovessels acts as a solid-phase regulator of angiogenesis, whose function varies depending on the concentration of its molecular components.

Gynecologic Oncology, 2010

The poor prognosis associated with epithelial ovarian cancer (EOC) is due to the lack of overt ea... more The poor prognosis associated with epithelial ovarian cancer (EOC) is due to the lack of overt early symptoms and the absence of reliable diagnostic screening methods. Since many tumors over express angiogenic regulators, the purpose of this study was to determine whether elevated levels of the angiogenic or angiostatic molecules vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), endostatin (ES), and angiostatin (AS) were elevated in plasma and urine from patients with EOC. VEGF, HGF, ES and AS were assayed by ELISA in samples from pilot cohort consisting of healthy women (N=48; pre-menopausal N=23, post-menopausal N=25), women with benign gynecological disease (N=54), patients with primary peritoneal cancer (PP) (N=2) and EOC (N=35). Wherever possible, parallel serum samples were measured for CA125 levels by ELISA. AS was the angioregulator that independently discriminated EOC patients from healthy individuals. Levels of urinary AS (uAS) from healthy individuals or women with benign gynecological disease averaged 21.4 ng/mL+/-3.7 and 41.5 ng/mL+/-8.8, respectively. In contrast, uAS averaged 115 ng/mL+/-39.2 and 276 ng/mL+/-45.8 from women with Stage I (N=6) and late stage (N=31) EOC, respectively. Furthermore, uAS was elevated in EOC patients regardless of tumor grade, stage, size, histological subtype, creatinine levels, menopausal status, or patient age, but appeared to complement CA125 measurements. Levels of AS are elevated in the urine of patients with EOC and may be of diagnostic and/or prognostic clinical importance. Further studies of uAS as a biomarker for EOC alone or in combination with other markers are warranted.

Current Pharmaceutical Design, 2009

Cancer Research, 2008

Hepatocellular carcinoma (HCC) is one of most malignant and aggressive human tumors. Transforming... more Hepatocellular carcinoma (HCC) is one of most malignant and aggressive human tumors. Transforming growth factor-β1 (TGF-β1) and its coreceptor CD105 have been shown to contribute to HCC malignant progression. TGF-β1 and CD105 have also been implicated in angiogenesis, but their role in the vascularization of HCC has not been investigated. To fill this gap, we studied the effect of TGF-β1 and CD105 on HCC-derived endothelium. By using immunomagnetic beads, we isolated and cultured endothelial cells (ECs) from HCC (HCC-EC) and adjacent nonneoplastic tissue (nNL-ECs) obtained from 24 liver biopsies. HCC and nNL biopsies were also analyzed by immunohistochemistry for the expression of CD105, TGF-β1, Ve-cadherin (Ve-cad), CD44, β-catenin, and E-cadherin. Compared with nNL-ECs, HCC-ECs had higher expression of CD105, enhanced spontaneous motility, and greater capacity to migrate in response to TGF-β1 (5 ng/mL), particularly in the presence of a fibronectin matrix. The chemotactic effect o...

Arteriosclerosis, Thrombosis, and Vascular Biology, 2011

Objective—The goal of this study was to define the role of tumor necrosis factor-α (TNFα) in the ... more Objective—The goal of this study was to define the role of tumor necrosis factor-α (TNFα) in the cascade of gene activation that regulates aortic angiogenesis in response to injury.Methods and Results—Angiogenesis was studied by culturing rat or mouse aortic rings in collagen gels. Gene expression was evaluated by quantitative reverse transcription–polymerase chain reaction, microarray analysis, immunocytochemistry, and ELISA. TNFα gene disruption and recombinant TNFα or blocking antibodies against vascular endothelial growth factor (VEGF) or TNF receptors were used to investigate TNFα-mediated angiogenic mechanisms. Resident aortic macrophages were depleted with liposomal clodronate. Angiogenesis was preceded by overexpression of TNFα and TNFα-inducible genes. Studies with isolated cells showed that macrophages were the main source of TNFα. Angiogenesis, VEGF production, and macrophage outgrowth were impaired by TNFα gene disruption and promoted by exogenous TNFα. Antibody-mediated...

Angiogenesis, 2007

Rat or mouse aortic rings produce angiogenic outgrowths in vitro through endogenous production of... more Rat or mouse aortic rings produce angiogenic outgrowths in vitro through endogenous production of growth factors and inflammatory cytokines. To further investigate this process in vivo, collagen-Gelfoam constructs containing aortic rings were implanted subcutaneously in syngeneic animals. Aortic rings stimulated a prominent angiogenic response characterized by peri- and intra-aortic accumulation of florid granulation tissue. Conversely, implants without rings elicited a non-specific inflammatory reaction without significant angiogenesis. The angiogenic response to the rings peaked at day 14 and was followed by regression of neovessels, which were mostly reabsorbed by day 28. Gene expression studies showed upregulated expression of angiogenic growth factors and cytokines in implants with rings. Tracking experiments with LacZ expressing ROSA26 transgenic mice demonstrated that both the aorta and the host contributed to the angiogenic response. These studies show that the angiogenic properties of the rodent aorta can be studied in the live animal under conditions that can be monitored and quantified. This in vivo assay can be used to study the molecular mechanisms by which the arterial wall and its proangiogenic cytokines regulate formation of granulation tissue during wound healing.

The American Journal of Pathology, 2007

Vasculogenesis, the formation of blood vessels in embryonic or fetal tissue mediated by immature ... more Vasculogenesis, the formation of blood vessels in embryonic or fetal tissue mediated by immature vascular cells (ie, angioblasts), is poorly understood. We report the identification of a population of vascular progenitor cells (hVPCs) in the human fetal aorta composed of undifferentiated mesenchymal cells that coexpress endothelial and myogenic markers. Under culture conditions that promoted cell differentiation, hVPCs gave rise to a mixed population of mature endothelial and mural cells when progenitor cells were stimulated with vascular endothelial growth factor-A or platelet-derived growth factor-␤␤. hVPCs grew as nonadherent cells and, when embedded in a three-dimensional collagen gel, reorganized into cohesive cellular cords that resembled mature vascular structures. hVPC-conditioned medium contained angiogenic substances (vascular endothelial growth factor-A and angiopoietin-2) and strongly stimulated the proliferation of endothelial cells. We also demonstrate the therapeutic efficacy of a small number of hVPCs transplanted into ischemic limb muscle of immunodeficient mice. hVPCs markedly improved neovascularization and inhibited the loss of endogenous endothelial cells and myocytes, thus ameliorating the clinical outcome from ischemia. We conclude that fetal aorta represents an important source for the investigation of the phenotypic and functional features of human vascular progenitor cells.

American Journal of Kidney Diseases, 2006