Placenta growth factor and vascular endothelial growth factor B expression in the hypoxic lung

Background

Chronic alveolar hypoxia, due to residence at high altitude or chronic obstructive lung diseases, leads to pulmonary hypertension, which may be further complicated by right heart failure, increasing morbidity and mortality. In the non-diseased lung, angiogenesis occurs in chronic hypoxia and may act in a protective, adaptive manner. To date, little is known about the behaviour of individual vascular endothelial growth factor (VEGF) family ligands in hypoxia-induced pulmonary angiogenesis. The aim of this study was to examine the expression of placenta growth factor (PlGF) and VEGFB during the development of hypoxic pulmonary angiogenesis and their functional effects on the pulmonary endothelium.

Methods

Male Sprague Dawley rats were exposed to conditions of normoxia (21% O₂) or hypoxia (10% O₂) for 1-21 days. Stereological analysis of vascular structure, real-time PCR analysis of vascular endothelial growth factor A (VEGFA), VEGFB, placenta growth factor (PlGF), VEGF receptor 1 (VEGFR1) and VEGFR2, immunohistochemistry and western blots were completed. The effects of VEGF ligands on human pulmonary microvascular endothelial cells were determined using a wound-healing assay.

Results

Typical vascular remodelling and angiogenesis were observed in the hypoxic lung. PlGF and VEGFB mRNA expression were significantly increased in the hypoxic lung. Immunohistochemical analysis showed reduced expression of VEGFB protein in hypoxia although PlGF protein was unchanged. The expression of VEGFA mRNA and protein was unchanged. In vitro PlGF at high concentration mimicked the wound-healing actions of VEGFA on pulmonary microvascular endothelial monolayers. Low concentrations of PlGF potentiated the wound-healing actions of VEGFA while higher concentrations of PlGF were without this effect. VEGFB inhibited the wound-healing actions of VEGFA while VEGFB and PlGF together were mutually antagonistic.

Conclusions

VEGFB and PlGF can either inhibit or potentiate the actions of VEGFA, depending on their relative concentrations, which change in the hypoxic lung. Thus their actions in vivo depend on their specific concentrations within the microenvironment of the alveolar wall during the course of adaptation to pulmonary hypoxia.     

Evolutionary analysis of human vascular endothelial growth factor, angiopoietin, and tyrosine endothelial kinase involved in angiogenesis and immunity

Human vascular endothelial growth factor (VEGF), angiopoietin (ANG) and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (TIE)-2 consist of a grouping of proteins that are involved in vascular homeostasis, vascular integrity and angiogenesis. There are nine proteins in the immediate VEGF family: VEGFA, VEGFB, VEGFC, VEGFD, VEGF-3, placental growth factor (PGF), VEGF receptor (VEGFR)-1, VEGFR-2, and VEGFR-1-related. They can be stimulated by cytokines to become involved in immune responses. By using in silico tools, we were able to identify several possible analogues or homologues of VEGF, ANG and TIE-2 in invertebrates. This is the first report to show that these proteins may be conserved through evolution. These proteins may have a role in vascular maintenance and immunity. In addition, since VEGF, ANG and TIE-2 have a role in mammalian immunity that is significantly influenced by cytokines, such as IL-1, this may indicate an interaction of the vascular system and the immune system over evolutionary time.     

Alterations in Cortical Morphology after Neonatal Stroke: Compensation in the Contralesional Hemisphere?

Although neonatal arterial ischemic stroke is now well‐studied, its complex consequences on long‐term cortical brain development has not yet been solved. In order to understand the brain development after focal early brain lesion, brain morphometry needs to be evaluated using structural parameters. In this work, our aim was to study and analyze the changes in morphometry of ipsi‐ and contralesional hemispheres in seven‐year‐old children following neonatal stroke. Therefore, we used surface‐based morphometry in order to examine the cortical thickness, surface area, cortical volume, and local gyrification index in two groups of children that suffered from neonatal stroke in the left (n = 19) and right hemispheres (n = 15) and a group of healthy controls (n = 30). Reduced cortical thickness, surface area, and cortical volumes were observed in the ipsilesional hemispheres for both groups in comparison with controls. For the group with left‐sided lesions, higher gyrification of the contralesional hemisphere was observed primarily in the occipital region along with higher surface area and cortical volume. As for the group with right‐sided lesions, higher gyrification was detected in two separate clusters also in the occipital lobe of the contralesional hemisphere, without a significant change in cortical thickness, surface area, or cortical volume. This is the first time that alterations of structural parameters are detected in the “healthy” hemisphere after unilateral neonatal stroke indicative of a compensatory phenomenon. Moreover, findings presented in this work suggest that lesion lateralization might have an influence on brain development and maturation.     

Possible role of VEGF in the progression of kidney disease in streptozotocin (STZ)-induced diabetic rats: effects of an ACE inhibitor and an angiotensin II receptor antagonist.

Two endothelium-derived factors, endothelin (ET), a vasoconstrictor, and vascular endothelial growth factor (VEGF), an angiogenic factor are thought to be involved in the pathogenesis of diabetic vascular complications. The aim of this study was to determine the effects of an angiotensin II type I (AT-1) receptor antagonist and an ACE inhibitor on the pathogenesis of VEGF and ET-1-mediated kidney disease in STZ-induced diabetic rats. Two days after STZ administration, diabetic rats were treated for 8 weeks with enalapril maleate, an ACE inhibitor, candesartan cilexetil, an AT-1 receptor antagonist, or saline. Urinary albumin and N-acetyl beta-D glucosaminidase (NAG) excretion as well as the VEGF protein content in the kidney were all found to be elevated in diabetic rats. Administration of enalapril maleate or candesartan cilexetil decreased the level of microalbuminuria and NAG excretion in diabetic rats. Administration of enalapril maleate also suppressed the elevated renal VEGF protein content in these animals while candesartan cilexetil treatment had no effect. Serum ET-1 and VEGF levels were unchanged by these treatments. These data support a role for AT-1 receptor antagonists and ACE inhibitors in the prevention of diabetic nephropathy, and suggest that the former may work by reducing renal VEGF levels.     

Time-dependent contribution of non neuronal cells to BDNF production after ischemic stroke in rats

Although brain-derived neurotrophic factor (BDNF) plays a central role in recovery after cerebral ischemia, little is known about cells involved in BDNF production after stroke. The present study testes the hypothesis that neurons are not the unique source of neosynthesized BDNF after stroke and that non neuronal-BDNF producing cells differ according to the delay after stroke induction. For this purpose, cellular localization of BDNF and BDNF content of each hemisphere were analysed in parallel before and after (4h, 24h and 8d) ischemic stroke in rats. Stroke of different severities was induced by embolization of the brain with variable number of calibrated microspheres allowing us to explore the association between BDNF production and neuronal death severity. The main results are that (a) unilateral stroke increased BDNF production in both hemispheres with a more intense and long-lasting effect in the lesioned hemisphere, (b) BDNF levels either of the lesioned or unlesioned hemispheres were not inversely correlated to neuronal death severity whatever the delay after stroke onset, (c) in the unlesioned hemisphere, stroke resulted in increased BDNF staining in neurons and ependymal cells (at 4h and 24h), (d) in the lesioned hemisphere, beside neurons and ependymal cells, microglial cells (at 24h), endothelial cells of cerebral arterioles (at 4h and 24h) and astrocytes (at 8d) exhibited a robust BDNF staining as well. Taken together, overall data suggest that non neuronal cells are able to produce substantial amount of BDNF after ischemic stroke and that more attention should be given to these cells in the design of strategies aimed at improving stroke recovery through BDNF-related mechanisms.     

Neutralization of endogenous vascular endothelial growth factor depletes primordial follicles in the mouse ovary

The regulation of early follicular growth and development involves a complex interaction of autocrine, paracrine, and endocrine signals. The ability of these factors to regulate follicle growth may depend in part on the extent of vascular delivery to and perfusion of the ovary. Vascular endothelial growth factor A (VEGFA) is a major regulator of vascular physiology in the ovary. VEGFA is produced in numerous ovarian compartments and likely plays a role in the regulation of all phases of follicular growth, from preantral through preovulatory. The aim of the present study was to further evaluate the role of VEGF in early follicle growth by neutralization of endogenous VEGF or VEGF receptors. Adult mice were injected systemically and prepubertal mice were injected directly under the ovarian bursa with antibodies designed to neutralize VEGF or block interaction with its receptors in the ovary. Both systemic and intrabursal injections of VEGF antibody significantly reduced the number of primordial follicles within 1-3 days after administration without affecting primary or secondary follicle numbers. Primordial follicle numbers were not different from control levels by 30 days after VEGFA antibody administration. Administration of antibodies to the kinase domain receptor (KDR), but not the FMS-like tyrosine receptor (FLT1), for VEGF also resulted in a significant decrease in primordial follicles. These data suggest that VEGF plays a vital role in the maintenance and growth of the primordial follicle pool.     

G Protein α Subunit 14 Mediates Fibroblast Growth Factor 2-Induced Cellular Responses in Human Endothelial Cells

During pregnancy, a tremendous increase in fetoplacental angiogenesis is associated with elevated blood flow. Aberrant fetoplacental vascular function may lead to pregnancy complications including pre-eclampsia. Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA) are crucial regulators of fetoplacental endothelial function. G protein α subunit 14 (GNA14), a member of Gαq/11 subfamily is involved in mediating hypertensive diseases and tumor vascularization. However, little is known about roles of GNA14 in mediating the FGF2- and VEGFA-induced fetoplacental endothelial function. Using human umbilical vein endothelial cells (HUVECs) cultured under physiological chronic low oxygen (3% O₂) as a cell model, we show that transfecting cells with adenovirus carrying GNA14 complementary DNA (cDNA; Ad-GNA14) increases (p < 0.05) protein expression of GNA14. GNA14 overexpression blocks (p < 0.05) FGF2-stimulated endothelial migration, whereas it enhances (p < 0.05) endothelial monolayer integrity (maximum increase of ~35% over the control at 24 hr) in response to FGF2. In contrast, GNA14 overexpression does not significantly alter VEGFA-stimulated cell migration, VEGFA-weakened cell monolayer integrity, and intracellular Ca⁺⁺ mobilization in response to adenosine triphosphate (ATP), FGF2, and VEGFA. GNA14 overexpression does not alter either FGF2- or VEGFA-induced phosphorylation of ERK1/2. However, GNA14 overexpression time-dependently elevates (p < 0.05) phosphorylation of phospholipase C-β3 (PLCβ3) at S1105 in response to FGF2, but not VEGFA. These data suggest that GNA14 distinctively mediates fetoplacental endothelial cell migration and permeability in response to FGF2 and VEGFA, possibly in part by altering activation of PLCβ3 under physiological chronic low oxygen.     

Valproic acid attenuates blood-spinal cord barrier disruption by inhibiting matrix metalloprotease-9 activity and improves functional recovery after spinal cord injury

In a retinal ischemic ex vivo model, we have reported protective effects of somatostatin (SRIF) receptor 2 (sst(2) ). As an ischemic condition not only causes cell death but also induces a vascular response, we asked whether vascular endothelial growth factor (VEGF) is altered in this model and whether its expression, release or localization are affected by sst(2) activation. Ex vivo retinas of wild-type (WT) and sst(1) KO mice (which over-express sst(2) ) were incubated in ischemic conditions with SRIF, octreotide (OCT) or a VEGF trap. Ischemia in WT retinas caused increase of VEGF release and decrease of VEGF mRNA. Both effects were counteracted by SRIF or OCT. VEGF immunoreactivity was in retinal neurons and scarcely in vessels. Ischemia caused a significant shift of VEGF immunoreactivity from neurons to vessels. The increase of vascular VEGF was reduced in sst(1) KO retinas and in WT retinas treated with SRIF or OCT. VEGF trap also limited this increase, demonstrating that vascular VEGF was of extracellular origin. Together, the data show a VEGF response to ischemia, in which VEGF released by damaged neurons reaches the retinal capillaries. The activation of sst(2) protects neurons from ischemic damage, thereby limiting VEGF release and the VEGF response.     

Transforming growth factor-β1 signalling triggers vascular endothelial growth factor resistance and monocyte dysfunction in type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) leads to monocyte dysfunction associated with atherogenesis and defective arteriogenesis. Transforming growth factor (TGF)-β1, placenta growth factor (PlGF)-1 and vascular endothelial growth factor (VEGF)A play important roles in atherogenesis and arteriogenesis. VEGF-receptor (VEGFR)-mediated monocyte migration is inhibited in T2DM (VEGFA resistance), while TGF-β1-induced monocyte migration is fully functional. Therefore, we hypothesize that TGF-β antagonises the VEGFA responses in human monocytes. We demonstrate that monocytes from T2DM patients have an increased migratory response towards low concentrations of TGF-β1, while PlGF-1/VEGFA responses are mitigated. Mechanistically, this is due to increased expression of type II TGF-β receptor in monocytes under high-glucose conditions and increased expression of soluble (s)VEGFR1, which is known to interfere with VEGFA signalling. VEGFA resistance in monocytes from T2DM patients can be rescued by either experimental down-regulation of TGF-β receptor expression in vitro or by functional blocking of TGF-β signalling using either a TGF-β receptor kinase inhibitor or a TGF-β neutralizing antibody. Our data demonstrate that both T2DM and high-glucose potentiate the TGF-β pathway. TGF-β signalling impairs VEGFR-mediated responses in T2DM monocytes and in this way contributes to mononuclear cell dysfunction, provide novel insights into T2DM vascular dysfunction.     

Robo4 maintains vessel integrity and inhibits angiogenesis by interacting with UNC5B

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature.     

Angiotensin II increases the permeability and PV-1 expression of endothelial cells

Angiotensin II (ANG II), the major effector molecule of the renin-angiotensin system (RAS), is a powerful vasoactive mediator associated with hypertension and renal failure. In this study the permeability changes and its morphological attributes in endothelial cells of human umbilical vein (HUVECs) were studied considering the potential regulatory role of ANG II. The effects of ANG II were compared with those of vascular endothelial growth factor (VEGF). Permeability was determined by 40 kDa FITC-Dextran and electrical impedance measurements. Plasmalemmal vesicle-1 (PV-1) mRNA levels were measured by PCR. Endothelial cell surface was studied by atomic force microscopy (AFM), and caveolae were visualized by transmission electron microscopy (TEM) in HUVEC monolayers. ANG II (10(-7) M), similarly to VEGF (100 ng/ml), increased the endothelial permeability parallel with an increase in the number of cell surface openings and caveolae. AT1 and VEGF-R2 receptor blockers (candesartan and ZM-323881, respectively) blunted these effects. ANG II and VEGF increased the expression of PV-1, which could be blocked by candesartan or ZM-323881 pretreatments and by the p38 mitogem-activated protein (MAP) kinase inhibitor SB-203580. Additionally, SB-203580 blocked the increase in endothelial permeability and the number of surface openings and caveolae. In conclusion, we have demonstrated that ANG II plays a role in regulation of permeability and formation of cell surface openings through AT1 receptor and PV-1 protein synthesis in a p38 MAP kinase-dependent manner in endothelial cells. The surface openings that increase in parallel with permeability may represent transcellular channels, caveolae, or both. These morphological and permeability changes may be involved in (patho-) physiological effects of ANG II.     

Newly identified biologically active and proteolysis-resistant VEGF-A isoform VEGF111 is induced by genotoxic agents

Ultraviolet B and genotoxic drugs induce the expression of a vascular endothelial growth factor A (VEGF-A) splice variant (VEGF111) encoded by exons 1–4 and 8 in many cultured cells. Although not detected in a series of normal human and mouse tissue, VEGF111 expression is induced in MCF-7 xenografts in nude mice upon treatment by camptothecin. The skipping of exons that contain proteolytic cleavage sites and extracellular matrix–binding domains makes VEGF111 diffusible and resistant to proteolysis. Recombinant VEGF111 activates VEGF receptor 2 (VEGF-R2) and extracellularly regulated kinase 1/2 in human umbilical vascular endothelial cells and porcine aortic endothelial cells expressing VEGF-R2. The mitogenic and chemotactic activity and VEGF111's ability to promote vascular network formation during embyonic stem cell differentiation are similar to those of VEGF121 and 165. Tumors in nude mice formed by HEK293 cells expressing VEGF111 develop a more widespread network of numerous small vessels in the peritumoral tissue than those expressing other isoforms. Its potent angiogenic activity and remarkable resistance to proteolysis makes VEGF111 a potential adverse factor during chemotherapy but a beneficial therapeutic tool for ischemic diseases.     

Role of ANG II in coronary capillary angiogenesis at the insulin-resistant stage of a NIDDM rat model

With the use of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of human non-insulin-dependent diabetes mellitus (NIDDM), we assessed whether ANG II is involved in coronary capillary angiogenesis at the insulin-resistant stage of NIDDM (20 wk of age). In OLETF rats, ANG II labeling and angiotensin type 1 (AT(1)) receptor expression in coronary vessels were increased more than in nondiabetic controls. A marked increase in vascular expression of vascular endothelial growth factor (VEGF) at both mRNA and protein levels was found in OLETF rats. The increased expression level of VEGF was associated with accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) activated by increased advanced glycation end products (AGEs). Morphometric analysis showed a significantly increased total coronary capillary density, which was a result of arterialization of the venular capillary portion in OLETF rats. Treatment of OLETF rats with candesartan, an AT(1) receptor blocker, inhibited vascular expressions of VEGF, HIF-1alpha, and AGEs, and ameliorated the morphometric changes. These results suggest a key role of ANG II in the pathogenesis of the coronary capillary remodeling in this NIDDM model.     

Effect of angiotensin II type 1 receptor antagonist on tumor growth and angiogenesis in a xenograft model of human bladder cancer

Several authors have investigated the antitumor activity of angiotensin II type 1 receptor (AT1R) antagonists, which are widely used as antihypertensive drugs. In this study, we evaluated the efficacy of the AT1R antagonist candesartan against bladder cancer. For the study in vitro, human bladder cancer cells (KU-19-19) were cultured with and without angiotensin II (A II) and candesartan, and cell viability and vascular endothelial growth factor (VEGF) secretion were analyzed. Also for the study in vivo, a tumor xenograft model was prepared in nude mice using KU-19-19 cells. Mice were administered candesartan daily by oral gavage, and paclitaxel via intravenous infusion. Microvessel density, VEGF expression, and apoptosis were investigated. Candesartan did not induce direct toxicity in KU-19-19 cells, but VEGF was significantly lower in candesartan-treated cells than in the A II-treated control cells. In mice, candesartan, paclitaxel and candesartan-paclitaxel significantly suppressed tumor growth to 46.0%, 35.8% and 17.3%, respectively, of the tumor volume in the control group, showing that combined treatment significantly inhibited tumor growth compared to the candesartan group. Microvessel density and VEGF were significantly decreased in the candesartan and candesartan-paclitaxel groups compared to the control group. The apoptotic index was significantly increased in the paclitaxel and candesartan-paclitaxel groups compared to the control and candesartan groups. In our experimental model, candesartan prevented bladder cancer growth by inhibiting angiogenesis. Furthermore, combined treatment with candesartan and paclitaxel enhanced paclitaxel-induced cytotoxicity. These results suggest that the AT1R antagonist candesartan may be a candidate for innovative therapy for bladder cancer.     

VEGFA Expression Is Inhibited by Arsenic Trioxide in HUVECs through the Upregulation of Ets-2 and miRNA-126

Arsenic trioxide (ATO) has been used to treat patients with acute promyelocytic leukemia. Recently, studies have shown that ATO can induce apoptosis in leukemic cells and blood vessel endothelial cells in a time- and dose-dependent manner through the inhibition of vascular endothelial growth factor A (VEGFA) production. VEGFA is a key factor in angiogenesis initiation. Targeted inhibition of VEGF or VEGFA expression can suppress angiogenesis; however, little is known about the mechanism by which ATO inhibits VEGFA expression. In this study, we investigated the role of miRNA-126 in the mechanism of action of ATO in human umbilical vein endothelial cells (HUVECs). ATO significantly decreased the viability and proliferation of HUVECs and decreased their migration at 48 h. Cell proliferation was inhibited by 50% (IC50) when 5.0 μmol/L ATO was used. ATO treatment induced miR-126 upregulation and HUVEC apoptosis. Transfection with a miR-126 mimic significantly downregulated VEGFA mRNA levels, and transfection with a miR-126 inhibitor significantly upregulated VEGFA mRNA levels. Finally, we showed that ATO treatment upregulated Ets-2 and miR-126 expression in HUVECs. These results demonstrate that ATO inhibits the growth of HUVECs and induces apoptosis by downregulating VEGFA. One mechanism by which this occurs is Ets-2 upregulation, which results in an increase in miR-126 levels and downregulation of VEGFA expression.     

Neuroprotective effects of VEGF administration after focal cerebral ischemia/reperfusion: dose response and time window

Administration of vascular endothelial growth factor (VEGF) has been shown to increase cerebral blood flow and reduce neurological damage after experimental ischemic brain injury. The purpose of this study was to examine the optimal dose and time window for the neuroprotective effect of VEGF when administrated after focal ischemia/reperfusion injury in rabbits. Focal cerebral ischemia/reperfusion was induced by the middle cerebral artery occlusion (MCAO) method. In a dose response experiment, low (1.25 ng/μL), middle (2.5 ng/μL) and high (5.0 ng/μL) doses of VEGF were administered 2h after MCAO by the route of perifocal region. The VEGF at a dose of middle (2.5 ng/μL) displayed excellent effects on neuroprotective efficacy for focal cerebral ischemia/reperfusion injury. In another experiment, 2.5 ng/μL VEGF was administered at times varying from 2 to 8h after MCAO. Infarct volume, water content and neurological deficits were significantly reduced when VEGF was given at 2 and 3h after injury. The protective effect was less when the same dose was given at the later times. Thus, the present findings indicated that VEGF reduced ischemic neuronal danger with a therapeutic time window within the first 3h of transient MCAO and may be useful in the treatment of acute ischemic stroke in humans.     

Vascular endothelial growth factor signaling in injured nerves underlies peripheral sensitization in neuropathic pain

Chronic neuroinflammation may be a critical component of intractable inflammatory diseases, including neuropathic pain. Because angiogenesis as a result of vascular endothelial growth factor (VEGF) signaling plays a pivotal role in inflammation, we focused on the mechanisms of VEGF‐regulated neuropathic pain in mice. The mRNA and protein expression of VEGFA were up‐regulated in the injured sciatic nerve after partial sciatic nerve ligation (PSL). VEGFA was localized to accumulated macrophages and neutrophils derived from bone marrow. Up‐regulation of VEGFA was mediated by histone H3 acetylation and trimethylation in its promoter region. VEGF receptors (VEGFR1 and VEGFR2) were localized to vascular endothelial cells or macrophages. By ex vivo fluorescence imaging and immunohistochemistry using DiI fluorescence, progression of angiogenesis was observed in the injured sciatic nerve after PSL. Perineural administration of pharmacological inhibitors of VEGFA and VEGFR tyrosine kinases prevented tactile allodynia and thermal hyperalgesia caused by PSL. Moreover, we determined the contribution of VEGF‐ and CXC‐chemokine receptor 4‐expressing angiogenic macrophages to neuropathic pain. Taken together, VEGFA is up‐regulated in injured peripheral nerves and participates in angiogenesis and prolonged pain behaviors through its receptors. We propose that VEGFA‐related components may underlie peripheral sensitization leading to neuropathic pain.

     

Early inhaled nitric oxide treatment decreases apoptosis of endothelial cells in neonatal rat lungs after vascular endothelial growth factor inhibition

Vascular endothelial growth factor (VEGF) receptor blockade impairs lung growth and decreases nitric oxide (NO) production in neonatal rat lungs. Inhaled NO (iNO) treatment after VEGF inhibition preserves lung growth in infant rats by unknown mechanisms. We hypothesized that neonatal VEGF inhibition disrupts lung growth by causing apoptosis in endothelial cells, which is attenuated by early iNO treatment. Three-day-old rats received SU-5416, an inhibitor of VEGF receptor, or its vehicle and were raised in room air with or without iNO (10 ppm). SU-5416 reduced alveolar counts and lung vessel density by 28% (P < 0.005) and 21% (P < 0.05), respectively, as early as at 7 days of age. SU-5416 increased lung active caspase-3 protein by 60% at 5 days of age (P < 0.05), which subsided by 7 days of age, suggesting a transient increase in lung apoptosis after VEGF blockade. Apoptosis primarily colocalized to lung vascular endothelial cells, and SU-5416 increased endothelial cell apoptotic index by eightfold at 5 days of age (P <0.0001). iNO treatment after SU-5416 prevented the increases in lung active caspase-3 and in endothelial cell apoptotic index. There was no difference in alveolar type 2 cell number between control and SU-5416-treated rats. We conclude that neonatal VEGF receptor inhibition causes transient apoptosis in pulmonary endothelium, which is followed by persistently impaired lung growth. Early iNO treatment after VEGF inhibition reduces endothelial cell apoptosis in neonatal lungs. We speculate that enhancing endothelial cell survival after lung injury may preserve neonatal lung growth in bronchopulmonary dysplasia.     

Favorable effect of VEGF gene transfer on ischemic peripheral neuropathy

Ischemic peripheral neuropathy is a frequent, irreversible complication of lower extremity vascular insufficiency. We investigated whether ischemic peripheral neuropathy could be prevented and/or reversed by gene transfer of an endothelial cell mitogen designed to promote therapeutic angiogenesis. Intramuscular gene transfer of naked DNA encoding vascular endothelial growth factor (VEGF) simultaneously with induction of hindlimb ischemia in rabbits abrogated the substantial decrease in motor and sensory nerve parameters, and nerve function recovered promptly. When gene transfer was administered 10 days after induction of ischemia, nerve function was restored earlier and/or recovered faster than in untreated rabbits. These findings are due in part to enhanced hindlimb perfusion. In addition, however, the demonstration of functional VEGF receptor expression by Schwann cells indicates a direct effect of VEGF on neural integrity as well. These findings thus constitute a new paradigm for the treatment of ischemic peripheral neuropathy.