Hypoxia-inducible factor 1 (HIF-1) is a dimeric transcription factor composed of HIF-1alpha and HIF-1beta subunits that plays an essential role in mammalian O2 homeostasis. In Hif1a-/- knockout mice, complete deficiency of HIF-1alpha resulted in cardiac and vascular malformations and embryonic lethality at E10.5. Between E8. 75 and E9.25 striking vascular regression and abnormal remodeling occurred in the cephalic region concomitant with marked mesenchymal cell death. Similar vascular defects were observed in HIF-1alpha- and VEGF-deficient embryos and VEGF mRNA expression was not induced by hypoxia in Hif1a-/- embryonic stem cells. Surprisingly, Hif1a-/- embryos demonstrated increased VEGF mRNA expression compared to wild-type embryos. In tissue culture cells, VEGF mRNA expression was induced by glucose deprivation independent of HIF-1alpha, providing a mechanism for increased VEGF mRNA expression in Hif1a-/- embryos, in which absence of adequate tissue perfusion resulted in both O2 and glucose deprivation. Rather than being associated with VEGF deficiency, the vascular defects in Hif1a-/- embryos were spatially correlated with cell death, the onset of which preceded vascular regression.

Neuropilin-1 (np-1) and neuropilin-2 (np-2) are receptors for axon guidance factors belonging to the class 3 semaphorins. np-1 also binds to the 165-amino acid heparin-binding form of VEGF (VEGF(165)) but not to the shorter VEGF(121) form, which lacks a heparin binding ability. We report that human umbilical vein-derived endothelial cells express the a17 and a22 splice forms of the np-2 receptor. Both np-2 forms bind VEGF(165) with high affinity in the presence of heparin (K(D) 1.3 x 10(-10) m) but not VEGF(121). np-2 also binds the heparin-binding form of placenta growth factor. These binding characteristics resemble those of np-1. VEGF(145) is a secreted heparin binding VEGF form that contains the peptide encoded by exon 6 of VEGF but not the peptide encoded by exon 7, which is present in VEGF(165). VEGF(145) binds to np-2 with high affinity (K(D) 7 x 10(-10) m). Surprisingly, VEGF(145) did not bind to np-1. Indeed, VEGF(145) does not bind to MDA-MB-231 breast cancer cells, which predominantly express np-1. By contrast, VEGF(145) binds to human umbilical vein-derived endothelial cells, which express both np-1 and np-2. The binding of VEGF(165) to porcine aortic endothelial cells expressing recombinant np-2 did not affect the proliferation or migration of the cells. Nevertheless, it is possible that VEGF-induced np-2-mediated signaling will take place only in the presence of other VEGF receptors such as VEGF receptor-1 or VEGF receptor-2.

Epithelial-mesenchymal transition (EMT) and hypoxia are considered as crucial events favouring invasion and metastasis of many cancer cells. In this study, different human neoplastic cell lines of epithelial origin were exposed to hypoxic conditions in order to investigate whether hypoxia per se may trigger EMT programme as well as to mechanistically elucidate signal transduction mechanisms involved. The following human cancer cell lines were used: HepG2 (from human hepatoblastoma), PANC-1 (from pancreatic carcinoma), HT-29 (from colon carcinoma) and MCF-7 (from breast carcinoma). Cancer cells were exposed to carefully controlled hypoxic conditions and investigated for EMT changes and signal transduction by using morphological, cell and molecular biology techniques. All cancer cells responded to hypoxia within 72 h by classic EMT changes (fibroblastoid phenotype, SNAIL and beta-catenin nuclear translocation and changes in E-cadherin) and by increased migration and invasiveness. This was involving very early inhibition of glycogen synthase kinase-3beta (GSK-3beta), early SNAIL translocation as well as later and long-lasting activation of Wnt/beta-catenin-signalling machinery. Experimental manipulation, including silencing of hypoxia-inducible factor (HIF)-1alpha and the specific inhibition of mitochondrial generation of reactive oxygen species (ROS), revealed that early EMT-related events induced by hypoxia (GSK-3beta inhibition and SNAIL translocation) were dependent on transient intracellular increased generation of ROS whereas late migration and invasiveness were sustained by HIF-1alpha- and vascular endothelial growth factor (VEGF)-dependent mechanisms. These findings indicate that in cancer cells, early redox mechanisms can switch on hypoxia-dependent EMT programme whereas increased invasiveness is sustained by late and HIF-1alpha-dependent release of VEGF.

Impaired angiogenesis and decreased vascular endothelial growth factor (VEGF) expression were recently documented in the remnant kidney (RK) model of progressive renal failure. VEGF (50 microg/kg, twice daily) was administered to RK rats between weeks 4 and 8 after surgery, and rats were euthanized at week 8 for histologic study. During the administration of VEGF (n = 7) or vehicle (n = 6), systemic BP was comparable in the two groups. VEGF treatment resulted in improved renal function and lower mortality rates, compared with the vehicle-treated group. Renal histologic analyses confirmed a 3.5-fold increase in glomerular endothelial cell proliferation (0.14 +/- 0.03 versus 0.04 +/- 0.02 proliferating endothelial cells/glomerulus, VEGF versus vehicle, P < 0.05), a twofold increase in peritubular capillary endothelial cell proliferation (1.60 +/- 0.30 versus 0.78 +/- 0.17 cells/mm(2), VEGF versus vehicle, P < 0.01), a threefold decrease in peritubular capillary rarefaction (P < 0.01), and a twofold increase in endothelial nitrix oxide synthase expression (P < 0.05) in the VEGF-treated group; an eightfold increase in urinary nitrate/nitrite levels (P < 0.05) was also noted. Although the difference in glomerulosclerosis scores did not reach statistical significance (0.67 +/- 0.42 versus 1.22 +/- 0.63, VEGF versus vehicle; range, 0 to 4; P = NS), VEGF-treated rats exhibited less interstitial collagen type III deposition (9.32 +/- 3.26 versus 17.45 +/- 7.50%, VEGF versus vehicle, P < 0.01) and reduced tubular epithelial cell injury, as manifested by osteopontin expression (5.57 +/- 1.60 versus 9.58 +/- 3.45%, VEGF versus vehicle, P < 0.01). In conclusion, VEGF treatment reduces fibrosis and stabilizes renal function in the RK model. The use of angiogenic factors may represent a new approach to the treatment of kidney disease.

Myocardial infarction and ischemic heart disease are the leading cause of death in the industrial world. Therapies employed for treating these diseases are aimed at promoting increased blood flow to cardiac tissue. Pharmacological induction of new coronary growth has recently been explored, however, clinical trials with known proangiogenic factors have been disappointing. To identify novel therapeutic targets, we have explored signaling pathways that govern embryonic coronary development. Using a combination of genetically engineered mice and an organ culture system, we identified novel roles for fibroblast growth factor (FGF) and Hedgehog (HH) signaling in coronary vascular development. We show that FGF signals promote coronary growth indirectly by signaling to the cardiomyoblast through redundant function of Fgfr1 and Fgfr2. Myocardial FGF signaling triggers a wave of HH activation that is essential for vascular endothelial growth factor (Vegf)-A, Vegf-B, Vegf-C, and angiopoietin-2 (Ang2) expression. We demonstrate that HH is necessary for coronary vascular development and activation of HH signaling is sufficient to promote coronary growth and to rescue coronary defects due to loss of FGF signaling. These studies implicate HH signaling as an essential regulator of coronary vascular development and as a potential therapeutic target for coronary neovascularization. Consistent with this, activation of HH signaling in the adult heart leads to an increase in coronary vessel density.

Angiogenesis is an essential biological process not only in embryogenesis but also in the progression of a variety of major diseases such as cancer, diabetes and inflammation. Vascular endothelial growth factor (VEGF) family and its receptor system has been shown to be the fundamental regulator in the cell signaling of angiogenesis. Other systems, Angiopoietin-Tie and EphrinB2-Eph4B etc. are also involved in and cooperate with VEGF system to establish the dynamic blood vessel structures. VEGF receptor belongs to PDGF receptor super-gene family, and carries seven Ig-domains in the extracellular region and a tyrosine kinase domain in the intracellular region. Three members of VEGF receptor family, Flt-1, KDR/Flk-1 and Flt-4, have unique characteristics in terms of the signal transduction, and regulate angiogenesis, lymphangiongenesis and vascular permeability. Further studies on VEGF-VEGF receptor system may significantly facilitate our understanding on the physiological as well as pathological vascular systems in the body and the development of new strategies to control and suppress the major diseases in humans.

In the RIP1-Tag2 mouse model of pancreatic islet carcinoma, angiogenesis is switched on in a discrete premalignant stage of tumor development, persisting thereafter. Signaling through VEGF receptor tyrosine kinases is a well-established component of angiogenic regulation. We show that five VEGF ligand genes are expressed in normal islets and throughout islet tumorigenesis. To begin dissecting their contributions, we produced an islet beta cell specific knockout of VEGF-A, resulting in islets with reduced vascularity but largely normal physiology. In RIP1-Tag2 mice wherein most oncogene-expressing cells had deleted the VEGF-A gene, both angiogenic switching and tumor growth were severely disrupted, as was the neovasculature. Thus, VEGF-A is crucial for angiogenesis in a prototypical model of carcinogenesis, whose loss is not readily compensated.

Vascular endothelial growth factor (VEGF) is a key angiogenic molecule that plays an important role in the growth and metastasis of many types of human cancer, including pancreatic adenocarcinoma. In this study, we explored the regulation of VEGF in human pancreatic cancer cells. Over 70% of the human pancreatic cancer cell lines studied in vitro secreted constitutively high levels of VEGF. High VEGF-secreting cells also generally expressed an elevated steady-state level of VEGF mRNA. Kinetic analysis revealed that the elevated steady-state level of VEGF mRNA was due to enhanced VEGF gene transcription and increased constitutive VEGF promoter activity. Deletive mutation analyses of the VEGF promoter revealed that the region from -109 to -38 bp was essential for constitutive VEGF promoter activity. Further deletion and point mutation analyses indicated that mutation of individual or all of the putative Sp1 binding sites reduced or eliminated the constitutive VEGF promoter activity and abrogated the differential activity of the promoter in high and low VEGF-expressing cells. Consistent with the constitutive VEGF transcription activation, a high level of constitutive Sp1 expression and activity was detected in pancreatic cancer cell lines and pancreatic cancer tissue specimens overexpressing VEGF. Collectively, our data demonstrated that constitutive Sp1 activation is essential for the differential overexpression of VEGF, which in turn plays an important role in the angiogenesis and progression of human pancreatic cancer.

Endostatin, a fragment of collagen XVIII, is a potent anti-angiogenic protein, but the molecular mechanism of its action is not yet clear. We examined the effects of endostatin on the biological and biochemical activities of vascular endothelial growth factor (VEGF). Endostatin blocked VEGF-induced tyrosine phosphorylation of KDR/Flk-1 and activation of ERK, p38 MAPK, and p125(FAK) in human umbilical vein endothelial cells. Endostatin also inhibited the binding of VEGF(165) to both endothelial cells and purified extracellular domain of KDR/Flk-1. Moreover, the binding of VEGF(121) to KDR/Flk-1 and VEGF(121)-stimulated ERK activation were blocked by endostatin. The direct interaction between endostatin and KDR/Flk-1 was confirmed by affinity chromatography. However, endostatin did not bind to VEGF. Our findings suggest that a direct interaction of endostatin with KDR/Flk-1 may be involved in the inhibitory function of endostatin toward VEGF actions and responsible for its potent anti-angiogenic and anti-tumor activities in vivo.

Intravenous administration of human bone marrow stromal cells (hMSCs) after middle cerebral artery occlusion (MCAo) in rats provides functional benefit. We tested the hypothesis that these functional benefits are derived in part from hMSC production of growth and trophic factors. Quantitative sandwich enzyme-linked immunosorbent assay (ELISA) of hMSCs cultured with normal and MCAo brain extracts were performed. hMSCs cultured in supernatant derived from ischemic brain extracts increased production of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). These neurotrophins and angiogenic growth factors increased in a post-ischemia time-dependent manner. The hMSC capacity to increase expression of growth and trophic factors may be the key to the benefit provided by transplanted hMSCs in the ischemic brain.

Affymetrix U133plus2 GeneChips were used to profile 59 head and neck squamous cell cancers. A hypoxia metagene was obtained by analysis of genes whose in vivo expression clustered with the expression of 10 well-known hypoxia-regulated genes (e.g., CA9, GLUT1, and VEGF). To minimize random aggregation, strongly correlated up-regulated genes appearing in >50% of clusters defined a signature comprising 99 genes, of which 27% were previously known to be hypoxia associated. The median RNA expression of the 99 genes in the signature was an independent prognostic factor for recurrence-free survival in a publicly available head and neck cancer data set, outdoing the original intrinsic classifier. In a published breast cancer series, the hypoxia signature was a significant prognostic factor for overall survival independent of clinicopathologic risk factors and a trained profile. The work highlights the validity and potential of using data from analysis of in vitro stress pathways for deriving a biological metagene/gene signature in vivo.

E7080 is an orally active inhibitor of multiple receptor tyrosine kinases including VEGF, FGF and SCF receptors. In this study, we show the inhibitory activity of E7080 against SCF-induced angiogenesis in vitro and tumor growth of SCF-producing human small cell lung carcinoma H146 cells in vivo. E7080 inhibits SCF-driven tube formation of HUVEC, which express SCF receptor, KIT at the IC(50) value of 5.2 nM and it was almost identical for VEGF-driven one (IC(50) = 5.1 nM). To assess the role of SCF/KIT signaling in tumor angiogenesis, we evaluated the effect of imatinib, a selective KIT kinase inhibitor, on tumor growth of H146 cells in nude mice. Imatinib did not show the potent antitumor activity in vitro (IC(50) = 2,200 nM), because H146 cells did not express KIT. However, oral administration of imatinib at 160 mg/kg clearly slowed tumor growth of H146 cells in nude mice, accompanied by decreased microvessel density. Oral administration of E7080 inhibited tumor growth of H146 cells at doses of 30 and 100 mg/kg in a dose-dependent manner and caused tumor regression at 100 mg/kg. While anti-VEGF antibody also slowed tumor growth, it did not cause tumor regression. These results indicate that KIT signaling has a role in tumor angiogenesis of SCF-producing H146 cells, and E7080 causes regression of H146 tumors as a result of antiangiogenic activity mediated by inhibition of both KIT and VEGF receptor signaling. E7080 may provide therapeutic benefits in the treatment of SCF-producing tumors.

Judah Folkman recognized that new blood vessel formation is important for tumor growth and proposed antiangiogenesis as a novel approach to cancer therapy. Discovery of vascular permeability factor VEGF-A as the primary tumor angiogenesis factor prompted the development of a number of drugs that targeted it or its receptors. These agents have often been successful in halting tumor angiogenesis and in regressing rapidly growing mouse tumors. However, results in human cancer have been less impressive. A number of reasons have been offered for the lack of greater success, and, here, we call attention to the heterogeneity of the tumor vasculature as an important issue. Human and mouse tumors are supplied by at least 6 well-defined blood vessel types that arise by both angiogenesis and arterio-venogenesis. All 6 types can be generated in mouse tissues by an adenoviral vector expressing VEGF-A(164). Once formed, 4 of the 6 types lose their VEGF-A dependency, and so their responsiveness to anti-VEGF/VEGF receptor therapy. If therapies directed against the vasculature are to have a greater impact on human cancer, targets other than VEGF and its receptors will need to be identified on these resistant tumor vessels.

Liver cancer remains a global health challenge, with an estimated incidence of >1 million cases by 2025. Hepatocellular carcinoma (HCC) is the most common form of liver cancer and accounts for ~90% of cases. Infection by hepatitis B virus and hepatitis C virus are the main risk factors for HCC development, although non-alcoholic steatohepatitis associated with metabolic syndrome or diabetes mellitus is becoming a more frequent risk factor in the West. Moreover, non-alcoholic steatohepatitis-associated HCC has a unique molecular pathogenesis. Approximately 25% of all HCCs present with potentially actionable mutations, which are yet to be translated into the clinical practice. Diagnosis based upon non-invasive criteria is currently challenged by the need for molecular information that requires tissue or liquid biopsies. The current major advancements have impacted the management of patients with advanced HCC. Six systemic therapies have been approved based on phase III trials (atezolizumab plus bevacizumab, sorafenib, lenvatinib, regorafenib, cabozantinib and ramucirumab) and three additional therapies have obtained accelerated FDA approval owing to evidence of efficacy. New trials are exploring combination therapies, including checkpoint inhibitors and tyrosine kinase inhibitors or anti-VEGF therapies, or even combinations of two immunotherapy regimens. The outcomes of these trials are expected to change the landscape of HCC management at all evolutionary stages.

Vascular endothelial growth factors (VEGFs) are a family of secreted polypeptides with a highly conserved receptor-binding cystine-knot structure similar to that of the platelet-derived growth factors. VEGF-A, the founding member of the family, is highly conserved between animals as evolutionarily distant as fish and mammals. In vertebrates, VEGFs act through a family of cognate receptor kinases in endothelial cells to stimulate blood-vessel formation. VEGF-A has important roles in mammalian vascular development and in diseases involving abnormal growth of blood vessels; other VEGFs are also involved in the development of lymphatic vessels and disease-related angiogenesis. Invertebrate homologs of VEGFs and VEGF receptors have been identified in fly, nematode and jellyfish, where they function in developmental cell migration and neurogenesis. The existence of VEGF-like molecules and their receptors in simple invertebrates without a vascular system indicates that this family of growth factors emerged at a very early stage in the evolution of multicellular organisms to mediate primordial developmental functions.

Store-operated Ca(2+) entry (SOCE) is the principal Ca(2+) entry mechanism in nonexcitable cells. Stromal-interaction molecule 1 (STIM1) is an endoplasmic reticulum Ca(2+) sensor that triggers SOCE activation. However, the role of STIM1 in regulating cancer progression remains controversial and its clinical relevance is unclear. Here we show that STIM1-dependent signaling is important for cervical cancer cell proliferation, migration, and angiogenesis. STIM1 overexpression in tumor tissue is noted in 71% cases of early-stage cervical cancer. In tumor tissues, the level of STIM1 expression is significantly associated with the risk of metastasis and survival. EGF-stimulated cancer cell migration requires STIM1 expression and EGF increases the interaction between STIM1 and Orai1 in juxta-membrane areas, and thus induces Ca(2+) influx. STIM1 involves the activation of Ca(2+)-regulated protease calpain, as well as Ca(2+)-regulated cytoplasmic kinase Pyk2, which regulate the focal-adhesion dynamics of migratory cervical cancer cells. Because of an increase of p21 protein levels and a decrease of Cdc25C protein levels, STIM1-silencing in cervical cancer cells significantly inhibits cell proliferation by arresting the cell cycle at the S and G2/M phases. STIM1 also regulates the production of VEGF in cervical cancer cells. Interference with STIM1 expression or blockade of SOCE activity inhibits tumor angiogenesis and growth in animal models, confirming the crucial role of STIM1-mediated Ca(2+) influx in aggravating tumor development in vivo. These results make STIM1-dependent signaling an attractive target for therapeutic intervention.

OBJECTIVE

To quantify the relationship between inflammation and angiogenesis in synovial tissue from patients with osteoarthritis (OA).

METHODS

Hematoxylin and eosin staining and histologic grading for inflammation were performed for 104 patients who met the American College of Rheumatology criteria for OA and had undergone total joint replacement or arthroscopy. A purposive sample of synovial specimens obtained from 70 patients was used for further analysis. Vascular endothelium, endothelial cell (EC) proliferating nuclei, macrophages, and vascular endothelial growth factor (VEGF) were detected by immunohistochemical analysis. Angiogenesis (EC proliferation, EC fractional area), macrophage fractional area, and VEGF immunoreactivity were measured using computer-assisted image analysis. Double immunofluorescence histochemical analysis was used to determine the cellular localization of VEGF. Radiographic scores for joint space narrowing and osteophyte formation in the knee were also assessed.

RESULTS

Synovial tissue samples from 32 (31%) of 104 patients with OA showed severe inflammation; thickened intimal lining and associated lymphoid aggregates were often observed. The EC fractional area, EC proliferation, and VEGF immunoreactivity all increased with increasing histologic inflammation grade and increasing macrophage fractional area. In the synovial intimal lining, VEGF immunoreactivity was localized to macrophages and increased with increasing EC fractional area and angiogenesis. No inflammation or angiogenic indices were significantly correlated with radiographic scores.

CONCLUSIONS

Inflammation and angiogenesis in the synovium are associated with OA. The angiogenic growth factor VEGF generated by the inflamed synovium may promote angiogenesis, thereby contributing to inflammation in OA.

Melanoma is the cancer with the highest increase in incidence, and transformation of radial growth to vertical growth (i.e., noninvasive to invasive) melanoma is required for invasive disease and metastasis. We have previously shown that p42/p44 MAP kinase is activated in radial growth melanoma, suggesting that further signaling events are required for vertical growth melanoma. The molecular events that accompany this transformation are not well understood. Akt, a signaling molecule downstream of PI3K, was introduced into the radial growth WM35 melanoma in order to test whether Akt overexpression is sufficient to accomplish this transformation. Overexpression of Akt led to upregulation of VEGF, increased production of superoxide ROS, and the switch to a more pronounced glycolytic metabolism. Subcutaneous implantation of WM35 cells overexpressing Akt led to rapidly growing tumors in vivo, while vector control cells did not form tumors. We demonstrated that Akt was associated with malignant transformation of melanoma through at least 2 mechanisms. First, Akt may stabilize cells with extensive mitochondrial DNA mutation, which can generate ROS. Second, Akt can induce expression of the ROS-generating enzyme NOX4. Akt thus serves as a molecular switch that increases angiogenesis and the generation of superoxide, fostering more aggressive tumor behavior. Targeting Akt and ROS may be of therapeutic importance in treatment of advanced melanoma.

The vascular beds supplying the retina may sustain injury as a result of underlying disease such as diabetes, and/or the interaction of genetic predisposition, environmental insults, and age. The vascular pathologic features observed in different intraocular vascular diseases can be categorized broadly as proliferation, exemplified by proliferative diabetic retinopathy, leakage such as macular edema secondary to retinal vein occlusion, or a combination of proliferation and leakage, as seen in neovascular age-related macular degeneration (AMD). The World Health Organization has identified diabetic retinopathy and AMD as priority eye diseases for the prevention of vision loss in developed countries. The pathologic transformations of the retinal vasculature seen in intraocular vascular disease are associated with increased expression of vascular endothelial growth factor A (VEGF), a potent endothelial-specific mitogen. Furthermore, in model systems, VEGF alone is sufficient to trigger intraocular neovascularization, and its inhibition is associated with functional and anatomic improvements in the affected eye. Therapeutic interventions with effect on VEGF include intraocular capture and neutralization by engineered antibodies or chimeric receptors, downregulation of its expression with steroids, or alleviation of retinal ischemia, a major stimulus for VEGF expression, with retinal ablation by laser treatment. Data from prospective randomized clinical trials indicate that VEGF inhibition is a potent therapeutic strategy for intraocular vascular disease. These findings are changing clinical practice and are stimuli for further study of the basic mechanisms controlling intraocular angiogenesis.

BACKGROUND

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Mitochondria can govern local concentrations of second messengers, such as reactive oxygen species (ROS), and mitochondrial translocation to discrete subcellular regions may contribute to this signaling function. Here, we report that exposure of pulmonary artery endothelial cells to hypoxia triggered a retrograde mitochondrial movement that required microtubules and the microtubule motor protein dynein and resulted in the perinuclear clustering of mitochondria. This subcellular redistribution of mitochondria was accompanied by the accumulation of ROS in the nucleus, which was attenuated by suppressing perinuclear clustering of mitochondria with nocodazole to destabilize microtubules or with small interfering RNA-mediated knockdown of dynein. Although suppression of perinuclear mitochondrial clustering did not affect the hypoxia-induced increase in the nuclear abundance of hypoxia-inducible factor 1α (HIF-1α) or the binding of HIF-1α to an oligonucleotide corresponding to a hypoxia response element (HRE), it eliminated oxidative modifications of the VEGF (vascular endothelial growth factor) promoter. Furthermore, suppression of perinuclear mitochondrial clustering reduced HIF-1α binding to the VEGF promoter and decreased VEGF mRNA accumulation. These findings support a model for hypoxia-induced transcriptional regulation in which perinuclear mitochondrial clustering results in ROS accumulation in the nucleus and causes oxidative base modifications in the VEGF HRE that are important for transcriptional complex assembly and VEGF mRNA expression.

Hypoxic preconditioning of stem cells and neural progenitor cells has been tested for promoting cell survival after transplantation. The present investigation examined the hypothesis that hypoxic preconditioning of bone marrow mesenchymal stem cells (BMSCs) could not only enhance their survival but also reinforce regenerative properties of these cells. BMSCs from eGFP engineered rats or pre-labeled with BrdU were pre-treated with normoxia (20% O(2), N-BMSCs) or sub-lethal hypoxia (0.5% O(2). H-BMSCs). The hypoxia exposure up-regulated HIF-1α and trophic/growth factors in BMSCs, including brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), vascular endothelial growth factor (VEGF) and its receptor FIK-1, erythropoietin (EPO) and its receptor EPOR, stromal derived factor-1 (SDF-1) and its CXC chemokine receptor 4 (CXCR4). Meanwhile, many pro-inflammatory cytokines/chemokines were down-regulated in H-BMSCs. N-BMSCs or H-BMSCs were intravenously injected into adult rats 24h after 90-min middle cerebral artery occlusion. Comparing to N-BMSCs, transplantation of H-BMSCs showed greater effect of suppressing microglia activity in the brain. Significantly more NeuN-positive and Glut1-positive cells were seen in the ischemic core and peri-infarct regions of the animals received H-BMSC transplantation than that received N-BMSCs. Some NeuN-positive and Glut-1-positive cells showed eGFP or BrdU immunoflourescent reactivity, suggesting differentiation from exogenous BMSCs into neuronal and vascular endothelial cells. In Rotarod test performed 15days after stroke, animals received H-BMSCs showed better locomotion recovery compared with stroke control and N-BMSC groups. We suggest that hypoxic preconditioning of transplanted cells is an effective means of promoting their regenerative capability and therapeutic potential for the treatment of ischemic stroke.

Most endocrine hormones are produced in tissues and organs with permeable microvessels that may provide an excess of hormones to be transported by the blood circulation to the distal target organ. Here, we investigate whether leptin, an endocrine hormone, induces the formation of vascular fenestrations and permeability, and we characterize its angiogenic property in the presence of other angiogenic factors. We provide evidence that leptin-induced new blood vessels are fenestrated. Under physiological conditions, capillary fenestrations are found in the leptin-producing adipose tissue in lean mice. In contrast, no vascular fenestrations were detected in the adipose tissue of leptin-deficient ob/ob mice. Thus, leptin plays a critical role in the maintenance and regulation of vascular fenestrations in the adipose tissue. Leptin induces a rapid vascular permeability response when administrated intradermally. Further, leptin synergistically stimulates angiogenesis with fibroblast growth factor (FGF)-2 and vascular endothelial growth factor (VEGF), the two most potent and commonly expressed angiogenic factors. These findings demonstrate that leptin has another new function-the increase of vascular permeability.

Bevacizumab (Avastin, Genentech) is a humanized monoclonal antibody targeting vascular endothelial growth factor (VEGF), a critical angiogenic factor involved in both physiological and pathological conditions. It has been recently approved by the US FDA as a first-line therapy for widespread metastatic colorectal cancer. This report is a detailed biological characterization of bevacizumab in a variety of in vitro models. It is shown that bevacizumab potently neutralizes VEGF and blocks its signal transduction through both the VEGFR-1 and VEGFR-2 receptors, as demonstrated by the inhibition of VEGF-induced cell proliferation, survival, permeability, nitric oxide production, as well as migration and tissue factor production. Although bevacizumab retains the ability to bind to human Fcgamma receptors and complement protein C1q, it does not demonstrate cell or complement-mediated cytotoxicity in either VEGF producing or targeting cells. Thus the mechanism of anti-tumor activity of bevacizumab is most likely due to its anti-angiogenesis effect through binding and neutralization of secreted VEGF.

BACKGROUND

Neovascularization is potentially important for the treatment of ischemic heart and limb disease. We reported that reactive oxygen species (ROS) derived from gp91phox (Nox2)-containing NAD(P)H oxidase are involved in angiogenesis in mouse sponge models as well as in vascular endothelial growth factor (VEGF) signaling in cultured endothelial cells. The role of gp91phox-derived ROS in neovascularization in response to tissue ischemia is unknown, however.

RESULTS

Here, we show that neovascularization in the ischemic hindlimb is significantly impaired in gp91phox-/- mice as compared with wild-type (WT) mice as evaluated by laser Doppler flow, capillary density, and microsphere measurements. In WT mice, inflammatory cell infiltration in the ischemic hindlimb was maximal at 3 days, whereas capillary formation was prominent at 7 days when inflammatory cells were no longer detectable. Increased O2*- production and gp91phox expression were present at both time points. The dihydroethidium staining of ischemic tissues indicates that O2*- is mainly produced from inflammatory cells at 3 days and from neovasculature at 7 days after operation. Relative to WT mice, ischemia-induced ROS production in gp91phox-/- mice at both 3 and 7 days was diminished, whereas VEGF expression was enhanced and the inflammatory response was unchanged. Infusion of the antioxidant ebselen into WT mice also significantly blocked the increase in blood flow recovery and capillary density after ischemia.

CONCLUSIONS

gp91phox-derived ROS play an important role in mediating neovascularization in response to tissue ischemia. NAD(P)H oxidases and their products are potential therapeutic targets for regulating angiogenesis in vivo.