Blood vessel formation in the tissue-engineered bone with the constitutively active form of HIF-1α mediated BMSCs

The successful clinical outcome of the implanted tissue-engineered bone is dependent on the establishment of a functional vascular network. A gene-enhanced tissue engineering represents a promising approach for vascularization. Our previous study indicated that hypoxia-inducible factor-1α (HIF-1α) can up-regulate the expression of vascular endothelial growth factor (VEGF) and stromal-derived factor 1 (SDF-1) in bone mesenchymal stem cells (BMSCs). The angiogenesis is a co-ordinated process that requires the participation of multiple angiogenic factors. To further explore the angiogenic effect of HIF-1α mediated stem cells, in this study, we systematically evaluated the function of HIF-1α in enhancing BMSCs angiogenesis in vitro and in vivo. A constitutively active form of HIF-1α (CA5) was inserted into a lentivirus vector and transduced into BMSCs, and its effect on vascularization and vascular remodeling was further evaluated in a rat critical-sized calvarial defects model with a gelatin sponge (GS) scaffold. The expression of the key angiogenic factors including VEGF, SDF-1, basic fibroblast growth factor (bFGF), placental growth factor (PLGF), angiopoietin 1 (ANGPT1), and stem cell factor (SCF) at both mRNAs and proteins levels in BMSCs were significantly enhanced by HIF-1α overexpression compared to the in vitro control group. In addition, HIF-1α-over expressing BMSCs showed dramatically improved blood vessel formation in the tissue-engineered bone as analyzed by photography of specimen, micro-CT, and histology. These data confirm the important role of HIF-1α in angiogenesis in tissue-engineered bone. Improved understanding of the mechanisms of angiogenesis may offer exciting therapeutic opportunities for vascularization, vascular remodeling, and bone defect repair using tissue engineering strategies in the future.     

Hypoxia-specific stabilization of HIF-1alpha by human papillomaviruses

Human papillomaviruses (HPV) are the causative agents of cervical cancer and have been shown to increase expression of pro-angiogenic factors from infected cells. Many angiogenic factors are regulated by hypoxia inducible factor 1α (HIF-1α). We investigated whether HPV31 affects the levels of HIF-1α under normal and hypoxic conditions. Our studies indicate that cells containing complete HPV31 genomes showed enhanced levels of HIF-1α upon treatment with the hypoxia mimic DFO, which resulted from protein stabilization and led to increased expression of some but not all HIF-1α target genes. Both HPV E6 and E7 were able independently to enhance induction of HIF-1α upon DFO treatment. Enhancement of HIF-1α stability was not restricted to high-risk HPV types, as HPV11, a low risk HPV type, mediated a similar effect. These findings shed light on mechanisms by which HPV contributes to angiogenesis both in benign cervical lesions and in cervical cancers.     

Brain microvasculature and hypoxia-related proteins in Alzheimer's disease

Alzheimer's disease (AD) is a progressive, neurodegenerative disease of increasing incidence. The pathologic processes that underlie this disorder are incompletely understood, however, hypoperfusion/hypoxia is thought to contribute to disease pathogenesis. Hypoxia inducible factor 1-alpha (HIF-1α), a key regulator of cellular responses to hypoxia, is elevated in the microcirculation of AD patients. Cerebral hypoxia is a potent stimulus for vascular activation and angiogenesis. Microvessels isolated from the brains of AD patients express a large number of angiogenic proteins. Despite considerable data in human tissues regarding vascular expression of hypoxia-related angiogenic proteins, there is little information regarding these proteins in the brain vasculature of transgenic AD mice. The objectives of this study were to determine expression of HIF-1α, angiogenic proteins, angiopoietin-2 (Ang-2), and matrix metalloproteinase 2 (MMP2), and survival/apoptotic proteins (Bcl-xL, caspase 3) in the cerebromicrovasculature of AD transgenic mice and to determine the direct effect of hypoxia on cerebral endothelial expression of these proteins in vitro. Cultured brain endothelial cells were subjected to hypoxia for 4-6 h and analyzed by western blot and immunofluorescence. Our results demonstrated that HIF-1α is induced in cultured brain endothelial cells exposed to hypoxia and that expression of Ang-2, MMP2 and caspase 3 was elevated and the anti-apoptotic protein Bcl-xL decreased. Brain sections from AD and control mice showed that HIF-1α, Ang-2, MMP2 and caspase 3 are elevated and Bcl-xL decreased in the microvasculature of AD mice. These data suggest the cerebromicrovasculature is an important target for the effects of hypoxia in the AD brain.     

Critical roles of cold shock domain protein A as an endogenous angiogenesis inhibitor in skeletal muscle

Angiogenesis is regulated by the local balance between angiogenic stimulators and inhibitors and is maintained by muscle-derived angiogenic factors in ischemic tissues. AIMS: Our objectives were to investigate the effect of cold shock domain protein A (CSDA) as an endogenous angiogenesis inhibitor and to develop a novel strategy of therapeutic angiogenesis by blocking CSDA expression. RESULTS: In human skeletal muscle cells, CSDA was upregulated during hypoxia when cells were damaged and apoptosis was induced. CSDA expression could repress the activity of hypoxia inducible factor-1α and nuclear factor κB, because CSDA can competitively bind the hypoxia response element and the nuclear factor κB-binding element. As a result, vascular endothelial growth factor-A, interleukin-6, and interleukin-8 secretions from skeletal muscle cells were decreased. Further, CSDA depletion increased the secretion level of these angiogenic factors. In a hindlimb ischemia model, transfer of short-hairpin RNA targeting CSDA ameliorated ischemia without direct transfer of angiogenic factors. In this ischemic tissue, vascular endothelial growth factor-A, interleukin-6, and CXCL2 protein levels were increased. INNOVATION AND CONCLUSION: CSDA appears to play a critical role as an endogenous angiogenesis inhibitor in skeletal muscle, and RNA interference targeting of CSDA is a promising gene therapy strategy for treating peripheral arterial disease.     

Angiogenesis in gliomas: biology and molecular pathophysiology

Glioblastoma multiforme (GBM) is characterized by exuberant angiogenesis, a key event in tumor growth and progression. The pathologic mechanisms driving this change and the biological behavior of gliomas remain unclear. One mechanism may involve cooption of native blood vessels by glioma cells inducing expression of angiopoietin-2 by endothelial cells. Subsequently, vascular apoptosis and involution leads to necrosis and hypoxia. This in turn induces angiogenesis that is associated with expression of hypoxia-inducible factor (HIF)-1alpha and vascular endothelial growth factor (VEGF) in perinecrotic pseudopalisading glioma cells. Here we review the molecular and cellular mechanisms implicated in HIF-1-dependent and HIF-1-independent glioma-associated angiogenesis. In GBMs, both tumor hypoxia and genetic alterations commonly occur and act together to induce the expression of HIF-1. The angiogenic response of the tumor to HIF-1 is mediated by HIF-1-regulated target genes leading to the upregulation of several proangiogenic factors such as VEGF and other adaptive response molecules. Understanding the roles of these regulatory processes in tumor neovascularization, tumor growth and progression, and resistance to therapy will ultimately lead to the development of improved antiangiogenic therapies for GBMs.     

MiR-199a inhibits the angiogenic potential of endometrial stromal cells under hypoxia by targeting HIF-1α/VEGF pathway

We previously reported that miR-199a suppressed the invasiveness of endometrial stromal cells (ESCs) by targeting IkappaB kinase beta (IKKβ). This study was to investigate the role of miR-199a in the angiogenic potential of ESCs under hypoxia. Forced overexpression of miR-199a in ESCs significantly attenuated its angiogenic potential under hypoxia. Moreover, miR-199a down-regulated the expression level of vascular endothelial growth factor-A (VEGF-A) in ESCs under hypoxic conditions. To delineate the mechanism by which miR-199a reduced VEGF-A production, further analysis of the target genes of miR-199a showed that miR-199a targeted both VEGF-A and Hypoxia-inducible factor (HIF)-1α in ESCs. Our findings indicate that miR-199a may attenuate the angiogenic potential of ESCs under hypoxia partly through HIF-1α/VEGF-A pathway suppression. Therefore, miR-199a may play pivotal roles in the pathogenesis of endometriosis and may become a potential therapeutic target of this disease.     

缺氧诱导因子-1在大鼠缺血性脑损伤中的双重作用

缺氧诱导因子-1(HIF-1)是低氧生理及病理过程中起作用的重要转录调控因子,由HIF-1α和HIF-1β两个亚单位组成。HIF-1调控的基因在能量代谢、红细胞生成、血管生成、血管扩张,细胞存活与凋亡中起一定作用。在大鼠缺血性脑损伤中,由于缺血时间和程度的不同,HIF-1对神经细胞具有保护和诱导凋亡的双重作用。它可以通过诱导靶基因如血管内皮细胞生长因子(VEGF)、促红细胞生成素(EPO)、葡萄糖转移蛋白-1(GLU-1)及葡萄糖合成酶的生成对缺血后脑细胞产生保护作用。然而,在严重缺氧条件下,HIF可以通过与肿瘤抑制蛋白p53结合、诱导bcl-2家族中的凋亡前基因BNIP3和NIX的表达以及促进诱导型一氧化氮合酶(iNOS)的生成而诱导细胞凋亡。特异性激活促进存活的基因或者抑制HIF-1的表达都可能成为临床治疗的策略。     

Constitutive expression of HIF-1alpha and HIF-2alpha in bone marrow stromal cells differentially promotes their proangiogenic properties

Bone marrow stromal cells (BMSCs) contain progenitors capable of participating in postnatal angiogenesis. Hypoxia-inducible factors (HIFs) mediate endothelial activation by driving the expression of multiple angiogenic factors. We explored the potential of HIF-1alpha and HIF-2alpha modification in BMSCs, as a tool to improve cell-based angiogenic therapy. BMSCs were retrovirally transduced to express stable forms of HIF-1alpha and HIF-2alpha. HIF-1alpha and, to a greater extent, HIF-2alpha overexpression promoted differentiation of BMSCs to the endothelial lineage, evident by CD31 and Tie-2 expression and improved adhesive properties. Whereas chemotaxis toward stromal-derived factor 1 was higher in both HIF-alpha-expressing BMSCs, enhanced migration toward vascular endothelial growth factor was found only following overexpression of HIF-2alpha, supported by a robust expression of its receptor, Flk-1. HIF-alpha expression was associated with upregulation of angiogenic proteins and improved tube formation. Cytokine arrays of endothelial cells stimulated by medium collected from HIF-alpha-expressing BMSCs revealed further angiogenic activation and improved adhesive capacity. Eventually, delivery of HIF-2alpha-transduced BMSCs induced a more robust angiogenic response, compared with sham-transduced or HIF-1alpha-transduced BMSCs in the corneal micropocket angiogenesis model. Our results support the use of HIF-alpha genes, particularly HIF-2alpha, to augment the efficacy of future cell-based therapy. Disclosure of potential conflicts of interest is found at the end of this article.     

Immunohistochemical investigation of the angiogenic proteins VEGF,HIF-1α and CD34 in invasive ductal carcinoma of the breast

The expression of prognostic markers in cancer has become important in diagnostic routine and research. A high mitotic rate compromises the individual cell access to oxygen and nutrients, due to reduced blood supply. Cells undertake adaptive measures such as vascular endothelial growth factor (VEGF), expressed under the control of hypoxia-inducible factor-1α (HIF-1α). CD34 is an endothelial marker which can show the presence and distribution of blood vessels. This study evaluated the presence and relative expression of VEGF, HIF-1α and CD34 using immunohistochemistry of 60 breast tumors and double staining, correlating the findings with clinical and pathological variables. High VEGF expression was correlated with low cell proliferation, lymph node-negative, smaller tumor size and patients not receiving hormone therapy. High HIF-1α expression predominated in younger (<50-year) patients, subjected to neo-adjuvant therapy and in p53-negative tumors. Absence of metastasis, radiotherapy or hormone treatment, and estrogen receptor (ER)-positive tumors showed high CD34 immunoreactivity. We suggest that the angiogenic factors VEGF, HIF-1α and CD34 are important in breast cancer progression and their abundance in breast tumors has prognostic and predictive value.     

Study of hypoxia-induced expression of HIF-1α in retina pigment epithelium

It was shown that the expression of HIF-1α in retinal pigment epithelium increased under hypoxic conditions. Eight hours after the start of hypoxic exposure, the expression of HIF-1α reached the peak and sustained after 24-hour hypoxia. However, the morphology of PRE cells began to change and the expression of HIF-1α decreased after long-term (48-hour) hypoxia. Hypoxia-induced increase in the level of HIF-1α in RPE. It can be an important step in choriodal neovascularization. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 3, pp. 293–297, March, 2007