Correlation between VEGF and HIF-1alpha expression in human oral squamous cell carcinoma

Understanding the development and progression of oral cancer is critical in the quest for successful therapeutic intervention. The hypoxic microenvironment present in human oral tumor in vivo may actively influence tumor growth and neovascularization. This study correlates expression of both VEGF and HIF-1alpha in normal keratinocytes and oral cancer cell lines and determine whether hypoxia played a role in VEGF and HIF-1alpha regulation. Three human oral cancer cell lines and three normal keratinocytes were exposed to both normoxia and hypoxia culture conditions. Northern and Western blot analysis were used to assess VEGF and HIF-1alpha expression in the different culture conditions. ELISA assays were performed to measure VEGF production in the different cell lines tested. Hypoxia upregulated VEGF and HIF-1alpha expression on both normal and oral cancer cell lines, with a statistically significant difference between normal and oral cancer cell lines. Pattern of hypoxia-induced VEGF mRNA level tightly followed the HIF-1alpha mRNA expression in the cell lines tested. These results suggest that hypoxia regulates both VEGF and HIF-1alpha expression in head and neck carcinoma cell lines, thus establishing a biochemical pathway between tumor hypoxia and neoangiogenesis in these aggressive neoplasms.     

In vivo and in vitro characteristic of HIF-1α and relative genes in ischemic femoral head necrosis

Background: Legg-Calvé-Perthes Disease (Perthes’ disease) is a childhood hip disorder initiated by ischemic necrosis of the growing femoral head. So far, the etiology and pathogenesis of Perthes’ disease is poorly understood. Materials and methods: Avascular osteonecrosis rat model was established to mimic the pathophysiological changes of femoral head necrosis. The chondrocytes of newborn Sprague-Dawley rats were isolated and cultured in hypoxic and normoxic condition. The expression characteristic of the hypoxia-inducible factor-1 alpha (HIF-1α) was evaluated both in vivo and in vitro models. Vascular endothelial growth factor (VEGF) and apoptotic genes in chondrocytes treated with normoxia and hypoxia were also studied. Results: HIF-1α expression increased greatly after ischemic operation and kept at relative high level in the arthromeningitis stage and declined in the stages of osteonecrosis and reconstruction. The HIF-1α mRNA levels of chondrocytes incubated at hypoxia were significantly higher than the cells treated with normoxia at 24 and 72 hours. Hypoxia inhibited VEGF expression; chondrocytes could oppose this inhibition manifested by the increasing of VEGF mRNA level after 72 hours hypoxia. The expression of apoptotic genes, Casp3, Casp8 and Casp9, elevated in chondrocytes after hypoxia with time differences. Conclusion: Hypoxia might be an etiological factor for femoral head necrosis, HIF-1α, VEGF as well as apoptotic genes participated the pathophysiological process of ischemic osteonecrosis.     

Chronic hypoxia enhances endothelin-1-induced intracellular calcium elevation in rat carotid body chemoreceptors and up-regulates ETA receptor expression

Endothelin-1 (ET-1) excites carotid body (CB) chemoreceptors and induces mitosis of the chemoreceptors in chronic hypoxia. The aim of the present study was to examine the hypothesis that up-regulation of both ETA receptor and endogenous ET-1 expression in CB chemoreceptors enhances the response of intracellular Ca2+ to ET-1 following adaptation to chronic hypoxia (10% inspired O2 for 3-4 weeks). Cytosolic free [Ca2+] ([Ca2+]i) in type-I (glomus) cells freshly dissociated from rat CBs was measured by spectrofluorometry. Application of exogenous ET-1 (1-100 nM) concentration-dependently elevated [Ca2+]i in the glomus cells. This response to ET-1 (100 nM) was 49% greater in the chronically hypoxic (CH) group. The ET-1 response was abolished completely by the ETA receptor antagonist BQ610 (1 microM), but not by the ETB antagonist BQ788 (1 microM). The transient [Ca2+]i elevation induced by caffeine (30 mM) in the normoxic group was similar to that in the CH group, suggesting no differences in the intracellular Ca2+ stores. In situ hybridization with a digoxigenin-labelled antisense ETA receptor mRNA oligonucleotide probe revealed very intense and ubiquitous specific expression of ETA receptors in the lobules of glomus cells in the CH group, whereas staining in normoxic controls was light. Immunohistochemical studies revealed intense cytoplasmic staining for ET-1-immunoreactivity in most of the cell clusters in glomera in the CBs of CH rats but was faint in normoxic CBs. These findings indicate increased expression of both the ETA receptor and ET-1 in CB chemoreceptors during chronic hypoxia. Taken together, our results suggest that the [Ca2+]i response to ET-1 in rat CB chemoreceptors is augmented by up-regulation of ETA receptors and ET-1 expression. The enhancement of the paracrine/autocrine effect of ET-1 on the chemoreceptors is consistent with an excitatory and mitogenic role of the ET-1 and ETA receptor in the CB during chronic hypoxia.     

Oxygen and life span: chronic hypoxia as a model for studying HIF-1alpha, VEGF and NOS during aging

To test if oxygen sensitive mechanisms are affected by hypoxia, we studied hypoxia inducible factor-1alpha (HIF-1alpha), vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS) expression by immunohistochemical analysis in young and old rat carotid bodies (CBs) using hypoxia as a model for modulating aging. Four groups of male age-matched Wistar rats (3 and 24 months) were used. Two groups were kept in room air, and two groups were kept under chronic intermittent hypoxia for 12 days. In aged carotid body and in hypoxia the increased expression of HIF-1alpha, VEGF, iNOS is less evident as compared to the young one. Electron microscopy sections showed a reduced mitochondrial number and area in the aged CBs and during hypoxia. Less responsiveness to hypoxia could be evidenced in the aged rats as compared to the young rats, suggesting an age dependency of the oxygen sensitive mechanisms.     

Hypoxia-inducible factor 1 alpha is essential for hypoxic p27 induction in endometrioid endometrial carcinoma

Hypoxia-inducible factor 1alpha (HIF-1alpha) plays an essential role in the adaptive response of cells to hypoxia. The cyclin-dependent kinase inhibitor p27(Kip1) is highly expressed in the normal endometrium but is lost during endometrial carcinogenesis. However, in high-grade cancers, p27 re-expression is observed. We analysed the role of HIF-1alpha in hypoxia-induced expression of p27 in vitro and in vivo in endometrial cancer. Paraffin-embedded specimens from endometrioid endometrial carcinoma (n = 39) were stained immunohistochemically for HIF-1alpha, p27, and Ki67. HEC1B, an endometrial carcinoma cell line, was cultured under normoxic or hypoxic conditions in the presence or absence of transiently expressed short hairpin RNAs targeting HIF-1alpha. Protein expression of p27 and HIF-1alpha was assessed by western blotting. Immunohistochemical staining revealed perinecrotic HIF-1alpha expression in 67% of the cases and p27 staining centrally in the tumour islands, mostly around necrosis, in 46% of the cases. In 50% of the tumours with perinecrotic HIF-1alpha expression, p27 and HIF-1alpha perinecrotic/central co-localization was observed. In these tumour sections, hypoxia-associated p27 expression showed less proliferation around necrosis. Analysis of cultured endometrial carcinoma cells demonstrated that p27 protein expression is induced by hypoxia. This induction was abrogated by transient knockdown of HIF-1alpha using RNAi. Furthermore, hypoxia induced cell cycle arrest in HEC1B cells. We conclude that, in endometrioid endometrial carcinoma, p27 re-expression by hypoxia is HIF-1alpha-dependent and leads to cell cycle arrest. This may contribute to the survival of cancer cells in hypoxic parts of the tumour.     

Expression of hypoxia-inducible factors in human endometrium and suppression of matrix metalloproteinases under hypoxic conditions do not support a major role for hypoxia in regulating tissue breakdown at menstruation

BACKGROUND: Classical studies in monkeys suggested that menstruation results from vasoconstriction, hypoxia and necrosis. The heterodimeric hypoxia-inducible factor (HIF) complex is critical in oxygen homeostasis via increased stability of HIF-1alpha/2alpha monomers, and these act as markers of hypoxia. We hypothesized that focal hypoxia in perimenstrual endometrium results in locally increased matrix metalloproteinases (MMP), leading to tissue destruction. METHODS: HIF-1alpha, HIF-2alpha and HIF-1beta were immunolocalized in cycling endometrium. Endometrial stromal cells were cultured under hypoxic and normoxic conditions and MMP measured by zymography and Western blots. RESULTS: HIF-1alpha and HIF-2alpha were detected in only some endometrial samples, and not confined to the perimenstrual tissue. Where present, they were primarily cytoplasmic, not nuclear. HIF-1beta was localized in epithelium, leukocytes and some decidual cells. Cultured endometrial stromal cells responded to hypoxia with increased cellular HIF-1alpha and secreted vascular endothelial growth factor. ProMMP-1 and proMMP-3 production was reduced in response to hypoxia regardless of the steroidal milieu (no added steroids, estrogen or estrogen plus progesterone). Active MMP-2 and membrane type 1 MMP but not proMMP-2 or proMMP-9 production were also inhibited by hypoxia. CONCLUSIONS: These results do not support a role for hypoxia in the focally increased production and activation of MMP observed prior to and during menstruation.     

The Relationship between HIF-2α and VEGF with Radiographic Severity in the Primary Osteoarthritic Knee

PurposeThe aim of this study was to determine the relationship of hypoxia-inducible factor-2 (HIF-2α) and vascular endothelial growth factor (VEGF) with radiographic severity in primary osteoarthritis (OA) of the knee. Expression of these two factors in cartilage samples from OA knee joints was examined at mRNA and protein levels.ResultsCartilage degeneration correlated with the radiographic severity grade. OA severity, determined using the Mankin scale, correlated positively with the KL grade (r=0.8790, p<0.01), and HIF-2α and VEGF levels with the radiographic severity of knee OA (r=0.7001, p<0.05; r=0.6647, p<0.05).ConclusionIn OA cartilage, HIF-2α and VEGF mRNA and protein levels were significantly and positively correlated. The expression of both factors correlated positively with the KL grade. HIF-2α and VEGF, therefore, may serve as biochemical markers as well as potential therapeutic targets in knee OA.     

Hypoxic inactivation of glycogen synthase kinase‐3β promotes gastric tumor growth and angiogenesis by facilitating hypoxia‐inducible factor‐1 signaling

Since the molecular mechanism of hypoxic adaptation in cancer cells is cell‐type specific, we investigated whether glycogen synthase kinase‐3β (GSK‐3β) activation is involved in hypoxia‐induced gastric tumor promotion. Stable gastric cancer cell lines (SNU‐638, SNU‐484, MKN1, and MKN45) were cultured under hypoxic conditions. Cells overexpressing wild‐type GSK‐3β (WT‐GSK‐3β) or kinase‐dead mutant of GSK‐3β (KD‐GSK‐3β) were generated and used for cell culture and animal studies. In cell culture experiments, hypoxia decreased GSK‐3β activation in gastric cancer cells. Cell viability and the expressions of HIF‐1α protein and VEGF mRNA in gastric cancer cells were higher in KD‐GSK‐3β transfectants than in WT‐GSK‐3β transfectants under hypoxic conditions, but not under normoxic conditions. Gastric cancer xenografts showed that tumor growth, microvessel area, HIF‐1α activation, and VEGF expression were higher in KD‐GSK‐3β tumors than in WT‐GSK‐3β tumors in vivo. In addition, the expression of hypoxia‐induced HIF‐1α protein was regulated by GSK‐3β at the translational level. Our data suggest that GSK‐3β is involved in hypoxic adaptation of gastric cancer cells as an inhibitory upstream regulator of the HIF‐1α/VEGF signaling pathway.     

Basic FGF augments hypoxia induced HIF-1-alpha expression and VEGF release in T47D breast cancer cells

AIM: Both hypoxia inducible factor 1 (HIF-1) and basic fibroblast growth factor (bFGF) play important roles in tumour angiogenesis. This study was designed to clarify the cooperative effect of these two mediators in induction of vascular endothelial cell growth factor (VEGF) release from breast cancer and probe possible mechanisms involved. METHODS: Release of VEGF from a breast cancer cell line (T47D) was quantitated by enzyme linked immunosorbent assay (ELISA). Expression of HIF-1 and ERK was assayed using Western blotting. Transient transfection and dual luciferase reporter assay were used to study HIF-1 transactivity. RESULTS: The data showed that hypoxia induced the expression of HIF-1alpha protein, the transactivity of HIF-1 and the release of VEGF. bFGF further augmented these hypoxic inductions. The PI3K pathway was required for these processes as demonstrated by application of PI3Kinase inhibitor (LY294002) or mutant construct transfections. In contrast, the MEK1 inhibitor PD98059 showed no effect on either activation of HIF-1 or VEGF release, which is in agreement with our finding that ERK1/2 was not activated by hypoxia. Under hypoxic conditions, bFGF activated the MEK1/ERK pathway. PD98059 blocked the activation of ERK1/2 and suppressed bFGF-induced HIF-1 transactivity, yet the protein expression of HIF-1alpha or VEGF release was not affected by PD98059. CONCLUSION: bFGF augments hypoxia induced VEGF release mainly through the PI3K pathway and partly depending on HIF-1 activity. Elucidation of this mechanism may provide a new target for anti-angiogenesis in cancer therapy.     

HIF-1α and VEGF expression correlates with thrombus remodeling in cases of intravascular papillary endothelial hyperplasia

Intravascular papillary endothelial hyperplasia (IPEH) is histopathologically characterized by endothelium-lined papillary structures encircling an acellular fibrin core. The process of IPEH pathogenesis is unclear. The purpose of our study was to identify histopathological and immunohistochemical characteristics of IPEH to better understand the pathogenesis of this disease. After reviewing microscopic and medical records from Kyungpook National University Hospital, we selected 16 cases of IPEH. Masson’s trichrome and immunohistochemical staining as well as hematoxylin-eosin staining for 16 cases of IPH were performed. Immunohistochemical studies included CD31, CD68, mast cell tryptase, hypoxia-inducible factor-1 (HIF-1α), and vascular endothelial growth factor (VEGF). Sections from all our cases showed three distinct histological regions including a papillary portion with hyalinized fibrous or fibroblastic cores, an area containing an unorganized thrombus, and organization area with an ingrowth of endothelial cells, myofibroblasts, and fibroblasts. In the organization area, HIF-1α-positive cells were identified in the loose connective tissue. Endothelial cells forming vascular channels were negative for HIF-1α while VEGF was highly expressed in both interstitial mononuclear and endothelial cells. In the papillary portion, the cellular cores were strongly positive for both HIF-1α and VEGF, but the acellular cores were negative. Our investigation confirmed that IPEH is a reactive lesion that incidentally arises during the organization process of older thrombi. It was also found that HIF-1α and VEGF expression was dependent on the thrombus remodeling stage in cases of IPEH.     

Experimental hypoxia and embryonic angiogenesis.

We examined the role of hypoxia and HIF factors in embryonic angiogenesis and correlated the degree of hypoxia with the level of HIF and VEGF expression and blood vessel formation. Quail eggs were incubated in normoxic and hypoxic (16% O(2)) conditions. Tissue hypoxia marker, pimonidazol hydrochloride, was applied in vivo for 1 hr and detected in sections with Hypoxyprobe-1 Ab. VEGF and HIF expression was detected by in situ hybridization. HIF-1alpha protein was detected in sections and by Western blot. Endothelial cells were visualized with QH-1 antibody. Hypoxic regions were detected even in normoxic control embryos, mainly in brain, neural tube, branchial arches, limb primordia, and mesonephros. The expression patterns of HIF-1alpha and HIF-1beta factors followed, in general, the Hypoxyprobe-1 marked regions. HIF-2alpha was predominantly expressed in endothelial cells. Diffuse VEGF expression was detected in hypoxic areas of neural tube, myocardium, digestive tube, and most prominently in mesonephros. Growing capillaries were directed to areas of VEGF positivity. Hypoxic regions in hypoxic embryos were larger and stained more intensely. VEGF and HIF-1 factors were proportionately elevated in Hypoxyprobe-1 marked regions without being expressed at new sites and were followed by increased angiogenesis. Our results demonstrate that normal embryonic vascular development involves the HIF-VEGF regulatory cascade. Experimentally increasing the level of hypoxia to a moderate level resulted in over-expression of HIF-1 factors and VEGF followed by an increase in the density of developing vessels. These data indicate that embryonic angiogenesis is responsive to environmental oxygen tension and, therefore, is not entirely genetically controlled.