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Vascular endothelial growth factor (VEGF) signaling is critical for tumor angiogenesis. However, therapies based on inhibition of VEGF receptors (VEGFRs) have shown modest results for patients with cancer. Surprisingly little is known about mechanisms underlying the regulation of VEGFR1 and VEGFR2 expression, the main targets of these drugs. Here, analysis of tissue microarrays revealed an inversely reciprocal pattern of VEGFR regulation in the endothelium of human squamous-cell carcinomas (high VEGFR1, low VEGFR2), as compared with the endothelium of control tissues (low VEGFR1, high VEGFR2). Mechanistic studies demonstrated that VEGF signals through the Akt/ERK pathway to inhibit constitutive ubiquitination and induce rapid VEGFR1 accumulation in endothelial cells. Surprisingly, VEGFR1 is primarily localized in the nucleus of endothelial cells. In contrast, VEGF signals through the JNK/c-Jun pathway to induce endocytosis, nuclear translocation, and downregulation of VEGFR2 via ubiquitination. VEGFR1 signaling is required for endothelial-cell survival, while VEGFR2 regulates capillary tube formation. Notably, the antiangiogenic effect of bevacizumab (anti-VEGF antibody) requires normalization of VEGFR1 and VEGFR2 levels in human squamous-cell carcinomas vascularized with human blood vessels in immunodeficient mice. Collectively, this work demonstrates that VEGF-induced angiogenesis requires inverse regulation of VEGFR1 and VEGFR2 in tumor-associated endothelial cells. [ABSTRACT FROM AUTHOR]

Expression of Bcl-xL correlates with the clinical outcomes of patients with cancer. While the role of Bcl-2 in angiogenesis is becoming increasingly evident, the function of Bcl-xL in angiogenesis is unclear. Here, we showed that epidermal growth factor (EGF) induces in vitro capillary sprouting and Bcl-xL expression in primary endothelial cells. Bcl-xL-transduced human dermal microvascular endothelial cells (HDMEC-Bcl-xL), but not empty vector control cells, spontaneously organize into capillary-like sprouts. Searching for a mechanism to explain these responses, we observed that Bcl-xL induced expression of the pro-angiogenic chemokines CXC ligand-1 (CXCL1) and CXC ligand-8 (CXCL8), and that blockade of CXC receptor-2 (CXCR2) signaling inhibited spontaneous sprouting of HDMEC-Bcl-xL. Bcl-xL led to Bcl-2 upregulation, but Bcl-2 did not upregulate Bcl-xL, suggesting the existence of a unidirectional crosstalk from Bcl-xL to Bcl-2. EGF and Bcl-xL activate the mitogen-activated protein kinase/ERK pathway resulting in upregulation of vascular endothelial growth factor (VEGF), a known inducer of Bcl-2 in endothelial cells. Inhibition of VEGF receptor signaling in HDMEC-Bcl-xL prevented Bcl-2 upregulation and demonstrated the function of a VEGF-mediated autocrine loop. Bcl-2 downregulation by RNAi blocked CXCL1 and CXCL8 expression downstream of Bcl-xL, and markedly decreased angiogenesis in vivo. We conclude that Bcl-xL functions as a pro-angiogenic signaling molecule controlling Bcl-2 and VEGF expression. These results emphasize a complex interplay between Bcl-2 family members beyond their classical roles in apoptosis.Cell Death and Differentiation (2007) 14, 1657–1666; doi:10.1038/sj.cdd.4402174; published online 15 June 2007 [ABSTRACT FROM AUTHOR]

The term apoptosis, also known as programmed cell death (PCD), was coined by developmental biologists a number of years ago to describe a form of cell death characterized by several unique morphological and biochemical features. Genetic studies of the round worm Caeneorhabditis elegans, a simple multicellular organism, first revealed apoptosis to be an integral part of the developmental program. Subsequently, the importance of apoptosis in higher organisms was demonstrated in several eukaryotic systems. In mammals, apoptosis is widespread during embryogenesis and in adult tissues. It is required for normal tissue homeostasis and for clonal selection in the immune system. In both developing and adult organisms, apoptosis plays a central role in reinforcing appropriate cellular patterns and in regulating cell number by eliminating cells that are harmful or no longer needed. It is becoming increasingly clear that disruption in the apoptosis pathway can contribute to the development of a number of developmental, inflammatory, degenerative, and neoplastic diseases. The effector arm of the apoptotic program includes members of the Bcl-2 gene family that function as either death agonists or death antagonists. These proteins participate in an elaborate genetically controlled biochemical pathway that functions to maintain tissue and organ homeostasis and serve as a critical defense mechanism to guard against malignant transformation. Cancer is the result of a series of genetic lesions that include activation of oncogenes and inactivation or loss of tumor suppressor genes. Several groups of investigators have observed that deregulated expression of oncogenes can subvert apoptotic pathways, resulting in prolonged cell survival. In pathological settings such as cancer, members of the Bcl-2 gene family are able to synergize with oncogenes and tumor suppressor genes to transform cells. In this review, we describe the process of apoptosis in mammalian cells and define the role and biochemical pathways through which the Bcl-2 gene family induce and/or protect cells from apoptosis. Last, we will discuss the evidence which suggests that alterations in this pathway may play a central role in tumorigenesis by allowing genetically damaged cells normally destined for elimination to persist, predisposing them to additional mutations and driving them to malignancy. [ABSTRACT FROM AUTHOR]

Dental pulp stem cells (DPSCs) constitute a unique group of cells endowed with multipotency, self-renewal, and capacity to regenerate the dental pulp tissue. While much has been learned about these cells in recent years, it is still unclear if each DPSC is multipotent or if unique sub-populations of DPSCs are "primed" to undergo specific differentiation paths. The purpose of the present study was to define whether a sub-population of DPSCs was uniquely primed to undergo vasculogenic differentiation. Permanent-tooth DPSCs or stem cells from human exfoliated deciduous teeth (SHED) were flow-sorted for vascular endothelial growth factor receptor 1 (VEGFR1) and exposed to vasculogenic differentiation medium, i.e., Microvascular-Endothelial-Cell-Growth-Medium-2-BulletKit™ supplemented with 50 ng/mL rhVEGF165 in the presence of 0 or 25 μg/mL anti-human VEGF antibody (bevacizumab; Genentech). In addition, sorted SHED (i.e., VEGFR1high or VEGFR1low) were seeded in biodegradable scaffolds and transplanted into the subcutaneous space of immunodeficient mice. Despite proliferating at a similar rate, VEGFR1high generated more in vitro sprouts than VEGFR1low cells (p < 0.05). Blockade of VEGF signaling with bevacizumab inhibited VEGFR1high-derived sprouts, demonstrating specificity of responses. Similarly, VEGFR1high SHED generated more blood vessels when transplanted into murine hosts than VEGFR1low cells (p < 0.05). Collectively, these data demonstrated that DPSCs contain a unique sub-population of cells defined by high VEGFR1 expression that are primed to differentiate into vascular endothelial cells. These data raise the possibility of purifying stem cells with high vasculogenic potential for regeneration of vascularized tissues or for vascular engineering in the treatment of ischemic conditions.

Background and Purpose: Increased angiogenesis in head and neck squamous cell carcinoma correlates to more aggressive tumors with increased morbidity. Because both elevated blood flow and high serum CXCL8 levels are correlated with increased angiogenesis, our objective was to see if elevated blood flow measured with CT perfusion correlated with CXCL8 levels, thereby helping to identify candidates for targeted therapies that inhibit the Bcl-2 proangiogenic pathway associated with CXCL8., Materials and Methods: Seven patients with locally recurrent or metastatic head and neck squamous cell carcinoma were enrolled in the trial. These patients underwent CT perfusion and the following parameters were measured: blood volume, blood flow, capillary permeability, and MTT; relative values were calculated by dividing by normal-appearing muscle. Serum was drawn for CXCL8 enzyme-linked immunosorbent assay analysis in these patients., Results: There was a significant positive correlation between the CXCL8 levels and relative blood flow (r = 0.94; P = .01). No correlation was found between CXCL8 and relative blood volume, relative capillary permeability, or relative MTT., Conclusions: Relative blood flow may be useful as a surrogate marker for elevated CXCL8 in patients with head and neck squamous cell cancer. Patients with elevated relative blood flow may benefit from treatment targeting the Bcl-2 proangiogenic pathways.

Anti-angiogenic therapies based on targeted disruption of the tumor microvascular network have been proposed for cancer treatment. Inhibitors of the endothelial cell pro-survival pathway mediated by VEGF were shown to activate caspases and cause microvascular regression, but the efficacy of this strategy can be hindered by the engagement of redundant survival pathways. Alternatively, if direct activation of an apical pro-apoptotic caspase is sufficient to disrupt microvessels in vivo, such a strategy could potentially override upstream endothelial cell survival inputs and disrupt tumor neovascular networks. Here, we fused caspase-9 to a mutated FKBP12 domain to express an inducible caspase-9 molecule (iCaspase-9) that can be activated by a cell-permeable dimerizer drug, and transduced this construct into primary endothelial cells. We found that drug-induced dimerization of iCaspase-9 is sufficient to activate endogenous caspase-3 and trigger apoptosis even when endothelial cells are treated with the pro-survival factors VEGF or bFGF. A single intraperitoneal injection of the dimerizer drug induced apoptosis of endothelial cells expressing iCaspase-9 and elimination of human microvessels engineered in immunodeficient mice. These results demonstrate that the activation of iCaspase-9 disrupts microvessels in vivo, and suggest a novel anti-angiogenic strategy based on the expression and controlled activation of an inducible death gene in neovascular endothelial cells.

Summary: Current model systems used to investigate angiogenesis in vivo rely on the interpretation of results obtained with nonhuman endothelial cells. Recent advances in tissue engineering and molecular biology suggest the possibility of engineering human microvessels in vivo. Here we show that human dermal microvascular endothelial cells (HDMEC) transplanted into severe combined immunodeficient (SCID) mice on biodegradable polymer matrices differentiate into functional human microvessels that anastomose with the mouse vasculature. HDMEC were stably transduced with Flag epitope or alkaline phosphatase to confirm the human origin of the microvessels. Endothelial cells appeared dispersed throughout the sponge 1 day after transplantation, became organized into empty tubular structures by Day 5, and differentiated into functional microvessels within 7 to 10 days. Human microvessels in SCID mice expressed the physiological markers of angiogenesis: CD31, CD34, vascular cellular adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1). Human endothelial cells became invested by perivascular smooth muscle alpha-actin-expressing mouse cells 21 days after implantation. This model was used previously to demonstrate that overexpression of the antiapoptotic protein Bcl-2 in HDMEC enhances neovascularization, and that apoptotic disruption of tumor microvessels is associated with apoptosis of surrounding tumor cells. The proposed SCID mouse model of human angiogenesis is ideally suited for the study of the physiology of microvessel development, pathologic neovascular responses such as tumor angiogenesis, and for the development and investigation of strategies designed to enhance the neovascularization of engineered human tissues and organs.

Vascular endothelial growth factor (VEGF) has been shown to be a potent mediator of angiogenesis that functions as a survival factor for endothelial cells by up-regulating Bcl-2 expression. We have recently reported that human dermal microvascular endothelial cells (HDMECs) seeded in biodegradable sponges and implanted into severe combined immunodeficient (SCID) mice organize into functional human microvessels that transport mouse blood cells. In this study, we implanted sponges seeded with OSCC-3 (oral squamous cell carcinoma) or SLK (Kaposi's sarcoma) together with endothelial cells into SCID mice to generate human tumors vascularized with human microvessels. This model system was used to examine the role of both endothelial cell Bcl-2 and the proangiogenic chemokine interleukin-8 (IL-8) on tumor growth and intratumoral microvascular density. Coimplantation of HDMECs overexpressing Bcl-2 (HDMEC-Bcl-2) and tumor cells resulted in a 3-fold enhancement of tumor growth when compared with the coimplantation of control HDMECs and tumor cells. This was associated with increased intratumoral microvascular density and enhanced endothelial cell survival. To determine whether the enhanced neovascularization mediated by Bcl-2 overexpression in endothelial cells was influenced by the synthesis of endogenous mediators of angiogenesis, we screened these cells for expression of VEGF, basic fibroblast growth factor (bFGF), and IL-8 by ELISA. HDMEC-Bcl-2 cells and VEGF-treated HDMECs exhibited a 15-fold and 4-fold increase, respectively, in the expression of the proangiogenic chemokine IL-8 in vitro, whereas the expression of VEGF and bFGF remained unchanged. Transfection of antisense Bcl-2 into HDMECs blocked VEGF-mediated induction of IL-8. Conditioned media from HDMEC-Bcl-2 induced proliferation and sprouting of endothelial cells in vitro and neovascularization in rat corneas. Anti-IL-8 antibody added to HDMEC-Bcl-2 conditioned media markedly reduced the potency of these responses. SCID mice bearing VEGF-producing tumor implants that were treated with anti-lL-8 antibody exhibited a 43% reduction in microvessel density and a 50% reduction in tumor weight compared with treatment with a nonspecific antibody. These results demonstrate that the up-regulation of Bcl-2 expression in endothelial cells that constitute tumor microvessels enhances intratumoral microvascular survival and density and accelerates tumor growth. Furthermore, endothelial cells that overexpress Bcl-2 have more angiogenic potential than control cells, and IL-8-neutralizing antibodies attenuate their angiogenic activity in vitro and in vivo.

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen and permeability factor that is potently angiogenic in vivo. We report here studies that suggest that VEGF potentiates angiogenesis in vivo and prolongs the survival of human dermal microvascular endothelial cells (HDMECs) in vitro by inducing expression of the anti-apoptotic protein Bcl-2. Growth-factor-enriched and serum-deficient cultures of HDMECs grown on collagen type I gels with VEGF exhibited a 4-fold and a 1.6-fold reduction, respectively, in the proportion of apoptotic cells. Enhanced HDMEC survival was associated with a dose-dependent increase in Bcl-2 expression and a decrease in the expression of the processed forms of the cysteine protease caspase-3. Cultures of HDMECs transduced with and overexpressing Bcl-2 and deprived of growth factors showed enhanced protection from apoptosis and exhibited a twofold increase in cell number and a fourfold increase in the number of capillary-like sprouts. HDMECs overexpressing Bcl-2 when incorporated into polylactic acid sponges and implanted into SCID mice exhibited a sustained fivefold increase in the number of microvessels and a fourfold decrease in the number of apoptotic cells when examined 7 and 14 days later. These results suggest that the angiogenic activity attributed to VEGF may be due in part to its ability to enhance endothelial cell survival by inducing expression of Bcl-2.