Increased vascular endothelial growth factor production in the lungs of rats with hypoxia-induced pulmonary hypertension

Vascular endothelial growth factor (VEGF) is a potent mitogenic and permeability factor targeting predominantly endothelial cells. At least two tyrosine kinase receptors, Flk-1 and Flt-1, mediate its action and are mostly expressed by endothelial cells. VEGF and VEGF receptor expression are upregulated by hypoxia in vivo and the role of VEGF in hypoxia-induced angiogenesis has been extensively studied in a variety of disease entities. Although VEGF and its receptors are abundantly expressed in the lung, their role in hypoxic pulmonary hypertension and the accompanying vascular remodeling are incompletely understood. We report in this in vivo study that hypoxia increases mRNA levels for both VEGF and Flk-1 in the rat lung. The kinetics of the hypoxic response differ between receptor and ligand: Flk-1 mRNA showed a biphasic response to hypoxia with a significant, but transient, rise in mRNA levels observed after 9-15 h of hypoxic exposure and the highest levels noted after 3 wk. In contrast, VEGF mRNA levels did not show a significant increase with acute hypoxia, but increased progressively after 1-3 wk of hypoxia. By in situ hybridization, VEGF mRNA was localized predominantly in alveolar epithelial cells with increased signal in the lungs of hypoxic animals compared with controls. Immunohistochemical staining with anti-VEGF antibodies localized VEGF peptide throughout the lung parenchyma and was increased in hypoxic compared with normoxic animals. Furthermore, hypoxic animals had significantly higher circulating VEGF concentrations compared with normoxic controls. Lung vascular permeability as measured by extravasation of Evans Blue dye was not significantly different between normoxic and hypoxic animals, although a tendency for increased permeability was seen in the hypoxic animals. These findings suggest a possible role for VEGF in the pulmonary response to hypoxia.     

Expression of platelet-derived growth factor and its receptors by two pre-B acute lymphocytic leukemia cell lines

Platelet-derived growth factors (PDGF) are potent regulators of cell proliferation. The three isoforms of PDGF AA, AB, and BB are encoded by two genes: PDGF A and PDGF B. The v-sis oncogene is homologous to the PDGF-B gene. v-sis can transform cells that express the appropriate PDGF receptors. Two different types of receptors, PDGF-alpha and PDGF-beta, also encoded by two genes, have been identified. We show that two cell lines. SMS-SB and NALM-6, both derived from pre-B-cell acute lymphocytic leukemias, express the PDGF-A chain gene, and one of them, SMS-SB, releases PDGF-A chains into the media. The SMS-SB cells also express the PDGF-beta receptor, whereas NALM-6 cells express the PDGF-alpha receptor and bind PDGF. This extends the possible targets for PDGF to the B-cell lineage lymphocytes.     

Lack of induction of substance P gene expression by hypoxia and absence of neurokinin 1-receptor mRNAs in the rat carotid body

Peripheral chemoreceptors are commonly thought to respond to hypoxia by releasing neurotransmitters from the type 1 cells of the carotid body; these molecules then bind to post-synaptic receptors on the carotid sinus nerve. The tachykinin substance P (SP) may act as an important neurotransmitter/neuromodulator in hypoxic chemotransmission in peripheral arterial chemoreceptors. In order to elucidate the role of SP in modulating hypoxic chemotransmission, we have used quantitative in situ hybridization histochemistry, to determine the effect of hypoxia on SP gene induction, and the localization of neurokinin 1 (NK-1) receptor mRNA in the carotid body and petrosal ganglia complex in rats at 21 days post-natal age. For comparison, we also determined: (1) the effect of hypoxia on tyrosine hydroxylase (TH) gene induction and (2) the localization of the mRNA encoding the D2-dopamine receptor. SP mRNA was not detected in the rat carotid body during normoxia and its expression was not induced after a 1 h of exposure to hypoxia (10% O2/90% N2), a stimulus that was sufficient to cause a significant increase (P < 0.01) in TH mRNA levels in the carotid body. Both SP and TH mRNAs were abundantly expressed in multiple cells in the petrosal and the jugular ganglia. However, these mRNAs were not co-localized and SP and TH mRNA levels were not affected by hypoxia in these ganglia. Although D2-dopamine receptor mRNA was abundantly expressed in the rat carotid body, we found no evidence of NK-1 receptor mRNA in the carotid body. In contrast, both NK-1 receptor mRNA and D2-dopamine receptor mRNA were present in petrosal ganglion cells. In the rat, SP does not appear to modulate hypoxic chemotransmission by being made in and released from type 1 cells in the carotid body, and neither does SP modulate the activity of type 1 cells by binding to NK-1 receptors on these cells.     

Hypoxia and vascular endothelial growth factor stimulate angiogenic integrin expression in bovine retinal microvascular endothelial cells

PURPOSE: Integrins alphavbeta3 and alphavbeta5 are cell-to-matrix adhesion molecules that have been reported to mediate vascular cell proliferation and migration. The authors investigated the regulation of expression of these angiogenic integrins by hypoxia and vascular endothelial growth factor (VEGF) in retinal microvascular endothelial cells in culture. METHODS: Cultured bovine retinal capillary endothelial cells were exposed to human recombinant VEGF under normoxic (95% air, 5% CO2) conditions to assess the effects of VEGF. Hypoxia studies were performed under lower oxygen concentration (0.5%-1.5% O2) induced by nitrogen replacement in constant 5% CO2 conditions. Integrin family mRNA and protein expression were assessed by northern blot analysis and immunoprecipitation. RESULTS: VEGF (25 ng/ml) increased integrin alphav, beta3, and 35 mRNA after 24 hours 6.1+/-0.8-fold (P < 0.001), 5.9+/-1.1-fold (P < 0.001), and 1.9+/-0.2-fold (P < 0.01), respectively. Similarly, hypoxia stimulated gene expression of integrin alphav and beta3 after 24 hours by 5.1+/-1.7-fold (P < 0.01) and 3.0+/-0.5-fold (P < 0.01), respectively, and integrin beta5 after 9 hours 1.4+/-0.2-fold (P < 0.05). This hypoxia-induced, integrin alphav mRNA elevation was inhibited significantly by anti-VEGF neutralizing antibody. Also, a conditioned medium from confluent endothelial cells maintained under hypoxic conditions for 24 hours produced a 7.1+/-1.1-fold increase (P < 0.001) in integrin alphav mRNA expression after 24 hours, which was reversed by anti-VEGF neutralizing antibody. Induction of integrin alphav by VEGF and hypoxia was confirmed in the protein level. CONCLUSIONS: These data suggest that hypoxia stimulates expression of vascular integrins alphavbeta3 and alphavbeta5 in retinal microvascular endothelial cells partially through autocrine-paracrine action of VEGF induced by the hypoxic state.     

Expression of platelet-derived growth factor (PDGF)-A, PDGF-B and the PDGF-alpha receptor, but not the PDGF-beta receptor, in human malignant melanoma in vivo

There has been considerable interest in the potential role of growth factors in the initiation and development of cutaneous malignant melanoma (CMM). Platelet-derived growth factor (PDGF) has been shown to be secreted by melanoma cell lines and by metastatic melanoma in vivo. PDGF also has been reported to stimulate the development of tumour stroma and new blood vessels. We studied the expression of PDGF and its receptors by both immunohistochemistry (IHC) and in situ hybridization (ISH) in primary and metastatic melanoma and in normal skin specimens. Cryostat sections were incubated with 35S-labelled riboprobes and antibodies for PDGF-AA, PDGF-alpha receptor, PDGF-BB and PDGF-beta receptor. Both primary and metastatic melanoma exhibited significant expression of PDGF-AA, PDGF-BB and PDGF-alpha receptor by both IHC and ISH, compared with only background expression in normal skin. We did not observe expression of PDGF-beta receptor in melanoma. Our results suggest that PDGF may function as an autocrine growth factor, as well as an angiogenesis factor, in CMM tumour development. This expression of the PDGF-alpha receptor rather than the beta receptor may be unique among solid tumours.     

Hypoxia stimulates insulin-like growth factor binding protein 1 (IGFBP-1) gene expression in HepG2 cells: a possible model for IGFBP-1 expression in fetal hypoxia

IGFBP-1 is elevated in fetuses with long-term, chronic hypoxia and intrauterine growth restriction. We investigated the hypothesis that hypoxia regulates IGFBP-1 in the human fetus in vivo and IGFBP-1 gene expression and protein in vitro. Umbilical artery IGFBP-1 levels (mean +/- SEM) from term babies with respiratory acidosis (acute hypoxia), normal babies, and those with mixed respiratory/metabolic acidosis (more profound and prolonged hypoxia) were measured using an immunoradiometric assay. IGFBP-1 levels were similar in normal (n = 12) and acutely hypoxic (n = 6) babies (189.1 +/- 71.8 vs. 175.8 +/- 45.9 ng /ml, respectively, P = 0.789). However, with more profound and prolonged hypoxia (n = 19), IGFBP-1 levels were markedly elevated (470.6 +/- 80.0 ng /ml, P = 0.044). To investigate IGFBP-1 regulation by hypoxia in vitro, HepG2 cells were incubated under hypoxia (pO2 = 2%) and normoxia (pO2 = 20%). IGFBP-1 protein and mRNA increased 8- and 12-fold, respectively, under hypoxic conditions. Hypoxia did not affect protein or mRNA levels of IGFBP-2 or -4. IGFBP-5 and -6 mRNAs, undetectable in control cells, were not induced by hypoxia, whereas minimally expressed IGFBP-3 mRNA increased twofold. Investigation into IGFBP-1 gene structure revealed three potential consensus sequences for the hypoxia response element (HRE) in the first intron. To investigate functionality, a 372-bp fragment of IGFBP-1 intron 1, containing putative HREs, was placed 5' to a heterologous hsp70 promoter in a plasmid using luciferase as a reporter gene. Under hypoxia, reporter gene activity increased up to 30-fold. Mutations in the middle HRE abolished reporter activity in response to hypoxia, suggesting that this HRE is functional in the IGFBP-1 hypoxia response. Cotransfection of HRE reporter genes with a constitutively expressing hypoxia-inducible factor 1 plasmid in HepG2 cells resulted in a fourfold induction of reporter activity, suggesting a role for hypoxia-inducible factor 1 in hypoxia induction of IGFBP-1 gene expression. These data support the hypothesis that hypoxia regulation of IGFBP-1 may be a mechanism operating in the human fetus to restrict insulin-like growth factor-mediated growth in utero under conditions of chronic hypoxia and limited substrate availability.     

Prolonged hypoxia increases vascular endothelial growth factor mRNA and protein in adult mouse brain

Brain hypoxia induces an increase in brain vascularity, presumably mediated by vascular endothelial growth factor (VEGF), but it is unclear whether VEGF is required to maintain the increase. In these studies, brain VEGF mRNA and protein levels were measured in adult mice kept in hypobaric chambers at 0.5 atm for 0, 0.5, 1, 2, 4, 7, and 21 days. Hypoxia was accompanied by a transient increase of VEGF mRNA expression: twofold by 0.5 day and a maximum of fivefold by 2 days; these were followed by a decrease at 4 days and a return to basal levels by 7-21 days. VEGF protein expression induced by hypoxia was bimodal, initially paralleling VEGF mRNA. There was an initial small increase at 12 h that reached a maximum by day 2, and, after a transient decrease on day 4, the protein expression increased again on day 7 before it returned to normoxic levels after 21 days. Thus, despite continued hypoxia, both VEGF mRNA and protein levels returned to basal after 7 days. These data suggest a metabolic negative-feedback system for VEGF expression during prolonged hypoxia in the brain.     

Hypoxia induces vascular endothelial growth factor gene and protein expression in cultured rat islet cells

The formation of new microvasculature by capillary sprouting at the site of islet transplantation is crucial for the long-term survival and function of the graft. Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties, may be a key factor in modulating the revascularization of islets after transplantation. In this study, we examined the gene expression of VEGF mRNA in three tumor cell lines and in isolated whole and dispersed rat islets in vitro by Northern blot hybridization in normoxic (5% CO2, 95% humidified air) and hypoxic (1% O2, 5% CO2, 94% N2) culture conditions. Increased expression of VEGF mRNA was observed in beta-TC3, RAW 264.7, and IC-21 tumor cell lines when subjected to hypoxia. With isolated whole islets in normoxic culture, a threefold increase in VEGF mRNA (P < 0.001) was seen at 48 h as compared with freshly isolated islets. This response was similar to the 3.8-fold increase observed with islets subjected to hypoxia. Dispersed rat islet cell clusters cultured on Matrigel for 24 h under hypoxic conditions showed a 3.4-fold increase (P < 0.01) in VEGF mRNA compared with those cultured in normoxia. This correlated with increased VEGF secretion as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies revealed the presence of increased expression of VEGF protein near the center of islets after 24 h of normoxic culture. Islet cell clusters on Matrigel showed intense cellular localization of VEGF in both beta-cells and non-beta-cells. These findings suggest that rat islet cells, when subjected to hypoxia during the first few days after transplantation, may act as a major source of VEGF, thereby initiating revascularization and maintaining the vascular permeability of the grafted islets.     

Hypoxia-mediated stimulation of carcinoma cell invasiveness via upregulation of urokinase receptor expression

Tumor hypoxia and high levels of expression of the urokinase-type plasminogen activator (uPA) receptor (uPAR) represent a poor clinical outcome for patients with various cancers. Here, we examined the effect of hypoxia on in vitro invasion of extracellular matrix and uPAR expression by human carcinoma cells. Compared with culture under 20% O2, culture for up to 24 hr under 1% or 4% O2 resulted in increased cell surface uPAR. However, the highest uPAR levels were observed in cells cultured under 1% O2. Culture of MDA-MB-231 breast carcinoma cells under hypoxia also resulted in increased uPAR mRNA levels. Furthermore, incubation with cobalt chloride or with an iron chelator also resulted in elevated uPAR expression, while presence of 30% carbon monoxide in the hypoxic atmosphere reduced the hypoxia-mediated uPAR mRNA upregulation. Increased uPAR expression was paralleled by higher cell-associated uPA levels and lower levels of secreted uPA as determined by gel zymography performed on cell extracts and culture-conditioned media. In addition, the in vitro invasiveness of MDA-MB-231 breast carcinoma cells was significantly higher when the invasion assay was performed under hypoxic conditions. This effect of hypoxia on invasion was abrogated by including in the assay a monoclonal, function-blocking anti-u PAR antibody or by the presence of 30% carbon monoxide in the hypoxic atmosphere. Our findings indicate that hypoxia stimulates carcinoma cell invasiveness by upregulating uPAR expression on the cell surface through a mechanism that requires a putative heme protein. Through a similar mechanism, hypoxia may stimulate tumor invasion and metastasis in vivo.     

Influence of applied tension and nitric oxide on responses to endothelins in rat pulmonary resistance arteries: effect of chronic hypoxia

1. The effect of basal tension (transmural tensions 235 +/- 29 mg wt (low tension: equivalent to approximately 16 mmHg) and 305 +/- 34 mg wt (high tension: equivalent to 35 mmHg)) on rat pulmonary resistance artery responses to endothelin-1 (ET-1) and the selective ET(B)-receptor agonist sarafotoxin S6c (S6c) were studied. The effects of nitric oxide synthase inhibition with N(omega)-nitro-L-arginine methylester (L-NAME, 100 microM) on ET receptor-induced responses, as well as vasodilator responses to acetylcholine (ACh) and S6c, were also investigated. Changes with development of pulmonary hypertension, induced by two weeks of chronic hypoxia, were determined. 2. Control rat preparations showed greatest sensitivity for ET-1 when put under low tension (pEC50: 8.1 +/- 0.1) compared with at the higher tension (pEC50: 7.7 +/- 0.1) and there were significant increases in maximum contractile responses to S6c (approximately 80%) and noradrenaline (approximately 60%) when put under high tension. 3. In control pulmonary resistance arteries, both ET-1 and S6c produced potent vasoconstrictor responses. S6c was 12 fold more potent than ET-1 in vessels set at low tension (S6c pEC50: 9.2 +/- 0.1) and 200 fold more potent than ET-1 when the vessels were set at high tension (S6c pEC50: 9.0 +/- 0.1). Chronic hypoxia did not change the potencies of ET-1 and S6c but did significantly increase the maximum contractile response to ET-1 by 60% (at low tension) and 130% (at high tension). 4. In control rat vessels, L-NAME itself caused small increases in vascular tone (5-8 mg wt tension) in 33-56% of vessels. In the chronic hypoxic rats, in vessels set at high tension, L-NAME-induced tone was evident in 88% of vessels and had increased to 26.9 +/- 6.6 mg wt tension. Vasodilatation to sodium nitroprusside, in non-preconstricted vessels, was small in control rat vessels (2-6 mg wt tension) but increased significantly to 22.5 +/- 8.0 mg wt tension in chronic hypoxic vessels set at the higher tensions. Together, these results indicate an increase in endogenous tone in the vessels from the chronic hypoxic rats which is normally attenuated by nitric oxide production. 5. L-NAME increased the sensitivity to S6c 10 fold (low tension) and 6 fold (high tension) only in chronic hypoxic rat pulmonary resistance arteries. It had no effect on responses to ET-1 in any vessel studied. 6. Vasodilatation of pre-contracted vessels by ACh was markedly greater in the pulmonary resistance arteries from the chronic hypoxic rats (pIC50: 7.12 +/- 0.19, maximum: 72.1 +/- 0.2.0%) compared to their age-matched controls (pIC50: 5.77 +/- 0.15, maximum: 28.2 +/- 2.0%). There was also a 2.5 fold increase in maximum vasodilatation induced by ACh. 7. These results demonstrate that control rat preparations showed greatest sensitivity for ET-1 when set at the lower tension, equivalent to the pressure expected in vivo (approximately 16 mmHg). Pulmonary hypertension due to chronic hypoxia potentiated the maximum response to ET-1. Pulmonary resistance arteries from control animals exhibited little endogenous tone, but exposure to chronic hypoxia increased endogenous inherent tone which is normally attenuated by nitric oxide. Endogenous nitric oxide production may increase in pulmonary resistance arteries from chronic hypoxic rats and attenuate contractile responses to ET(B2) receptor stimulation. Relaxation to ACh was increased in pulmonary resistance arteries from chronic hypoxic rats.     

Localization and sex steroid regulation of androgen receptor gene expression in rhesus monkey uterus

OBJECTIVE: To characterize the cellular sites and hormonal regulation of uterine androgen receptor gene expression in the monkey. METHODS: Ovariectomized rhesus monkeys (five in each group) were treated with placebo (the control group), estradiol (E2), E2 plus progesterone, or E2 plus testosterone by sustained-release pellets administered subcutaneously. After 3 days of treatment, uteri were removed and uterine sections were analyzed by in situ hybridization for androgen receptor messenger RNA (mRNA). RESULTS: Androgen receptor mRNA was detected in endometrial stromal cells and myometrial smooth muscle cells, with lesser expression in endometrial epithelial cells. Both E2 and E2 plus progesterone treatment doubled androgen receptor mRNA levels in stromal cells (P < .01), whereas E2 plus testosterone treatment increased stromal androgen receptor mRNA levels by about five-fold (P < .001) compared with placebo treatment. In the endometrial epithelium, E2 alone did not increase androgen receptor mRNA levels significantly. However, the E2 plus progesterone and E2 plus testosterone treatments increased epithelial androgen receptor mRNA levels by 4.3 and 5 times, respectively (P = .008 and P < .002, respectively). Androgen receptor mRNA was distributed homogeneously in smooth muscle cells across the myometrium. Estradiol treatment alone did not increase myometrial androgen receptor mRNA levels significantly, but the E2 plus progesterone and E2 plus testosterone treatments increased myometrial androgen receptor mRNA levels by 1.8 and 2 times, respectively (P = .001 and P < .001, respectively). CONCLUSION: Androgen receptor gene expression was detected in all uterine cell compartments where it was subject to significant sex steroid regulation. The fact that androgen receptor mRNA levels were consistently up-regulated by a combined E2 plus testosterone treatment while E2 treatment alone had little or no effect shows that a collaborative action of E2 and testosterone enhances androgen receptor expression in the monkey uterus.     

Expression of TGF-beta 1, PDGF and IGF-1 mRNA in lung of bleomycin-A5-induced pulmonary fibrosis in rats

OBJECTIVE: To investigate the influence of alveolar macrophages (AMs), fibroblasts and interstitial cells on development of lung fibrosis, and the interactions among TGF-beta 1 PDGF and IGF-1 and these cytokines-effects on lung fibrosis. MATERIAL AND METHODS: Expressions of TGF-beta 1, PDGF and IGF-1 mRNA in the lung cells and lung tissues in different stages of Bleomycin-A5-induced pulmonary fibrosis in rats were studied through Northern hybridization. RESULTS: The expressions of TGF-beta 1 and PDGF mRNA reached their peaks in AMs of pulmonary fibrosis in rats on the 7th day after Bleomycin-A5 instillation. It was similar with that in the lung tissues. IGF-1 mRNA remained relatively stable in AMs during the course. PDGF and IGF-1 mRNA increased gradually in fibroblasts, and reached the highest expressions in the interstitial cells. There was almost no TGF-beta 1 mRNA expression in all groups of fibroblasts. CONCLUSIONS: AMs are the main sources of TGF-beta 1 and PDGF in the lung tissues with fibrosis induced by Bleomycin-A5 AMs are activated in the first weekend and secrete TGF-beta 1 and PDGF to promote fibroblasts proliferation and fibrosis. As fibrosis developed, fibroblasts have established PDGF and IGF-1 autocrine and these three cytokines paracrine nets combined with the interstitial cells to promote lung fibrosis.