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Biomedical subjects

N Ferrara

Publications and source records attributed to N Ferrara.

At least 91 records · Page 5Linked to original sources
PubMed1996

Complete inhibition of angiogenesis and growth of microtumors by anti-vascular endothelial growth factor neutralizing antibody: novel concepts of angiostatic therapy from intravital videomicroscopy.

In the present study, we evaluated the effects of a neutralizing antivascular endothelial growth factor (anti-VEGF) antibody on angiogenesis and growth of tumor spheroids using an intravital microscopic technique permitting noninvasive, in vivo and in situ study of tumor angiogenesis and tumor growth in conscious mice. Tumor spheroids of the human rhabdomyosarcoma cell line A673, with a diameter between 600 and 1000 microns, were implanted in dorsal skinfold chambers inserted on Beige nude/xid mice. Tumor cells were prelabeled with a fluorescent vital dye [(5-(and-6)-((4-chloromethyl)benzoyl)amino)tetramethylrhodamine], which allowed estimation of the growth of the implanted tumor spheroids. Treatment (i.p.) with the monoclonal antibody A4.6.1, specific for VEGF, completely inhibited neovascularization of the microtumors and suppressed their growth to the extent that tumors implanted in treated animals leveled off at a volume less than 1 mm3, i.e., the anti-VEGF antibody dramatically changed the growth characteristics of the tumor line from being a rapidly growing malignancy to a dormant microcolony.

Animals↗
PubMed1996

Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene.

Angiogenesis is required for a wide variety of physiological and pathological processes. The endothelial cell-specific mitogen vascular endothelial growth factor (VEGF) is a major mediator of pathological angiogenesis. Also, the expression of VEGF and its two receptors, Flt-1 and Flk-1/KDR, is related to the formation of blood vessels in mouse and rat embryos. Mice homozygous for mutations that inactivate either receptor die in utero between days 8.5 and 9.5. However, ligand(s) other than VEGF might activate such receptors. To assess the role of VEGF directly, we disrupted the VEGF gene in embryonic stem cells. Here we report the unexpected finding that loss of a single VEGF allele is lethal in the mouse embryo between days 11 and 12. Angiogenesis and blood-island formation were impaired, resulting in several developmental anomalies. Furthermore, VEGF-null embryonic stem cells exhibit a dramatically reduced ability to form tumours in nude mice.

Alleles↗
PubMed1996

The carboxyl-terminal domain (111-165) of vascular endothelial growth factor is critical for its mitogenic potency.

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for endothelial cells. VEGF is synthesized and secreted by many differentiated cells in response to a variety of stimuli including hypoxia. VEGF is expressed in a variety of tissues as multiple homodimeric forms (121, 165, 189, and 206 amino acids/monomer) resulting from alternative RNA splicing. VEGF121 is a soluble mitogen that does not bind heparin; the longer forms of VEGF bind heparin with progressively higher affinity. The higher molecular weight forms of VEGF can be cleaved by plasmin to release a diffusible form(s) of VEGF. We characterized the proteolysis of VEGF by plasmin and other proteases. Thrombin, elastase, and collagenase did not cleave VEGF, whereas trypsin generated a series of smaller fragments. The isolated plasmin fragments of VEGF were compared with respect to heparin binding, interaction with soluble VEGF receptors, and ability to promote endothelial cell mitogenesis. Plasmin yields two fragments of VEGF as indicated by reverse phase high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis: an amino-terminal homodimeric protein containing receptor binding determinants and a carboxyl-terminal polypeptide which bound heparin. Amino-terminal sequencing of the carboxyl-terminal peptide identified the plasmin cleavage site as Arg110-Ala111. A heterodimeric form of VEGF165/110, was isolated from partial plasmin digests of VEGF165. The carboxyl-terminal polypeptide (111-165) displayed no affinity for soluble kinase domain region (KDR) or Fms-like tyrosine kinase (FLT-1) receptors. The various isoforms of VEGF (165, 165/110, and 121) bound soluble kinase domain region receptor with similar affinity (approximately 30 pM). In contrast, soluble FLT-1 receptor differentiated VEGF isoforms (165, 165/110, 110, and 121) with apparent affinities of 10, 30, 120, and 200 pM, respectively. Endothelial cell mitogenic potencies of VEGF110 and VEGF121 were decreased more than 100-fold compared to that of VEGF165. The present findings indicate that removal of the carboxyl-terminal domain, whether it is due to alternative splicing of mRNA or to proteolysis, is associated with a significant loss in bioactivity.

Adrenal Cortex↗
PubMed1996

Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis.

Vascular endothelial growth factor (VEGF) expression in various cell types is induced by hypoxia and other stimuli. VEGF mediates endothelial cell proliferation, angiogenesis, vascular growth, and vascular permeability via the endothelial cell receptors, kinase insert domain-containing receptor (KDR)/fetal liver kinase 1 (Flk-1) and FLT-1. Alanine-scanning mutagenesis was used to identify a positively charged surface in VEGF that mediates binding to KDR/Flk-1. Arg82, Lys84 and His86, located in a hairpin loop, were found to be critical for binding KDR/Flk-1, while negatively charged residues, Asp63, Glu64, and Glu67, were associated with FLT-1 binding. A VEGF model based on PDGFb indicated these positively and negatively charged regions are distal in the monomer but are spatially close in the dimer. Mutations within the KDR site had minimal effect on FLT-1 binding, and mutants deficient in FLT-1 binding did not affect KDR binding. Endothelial cell mitogenesis was abolished in mutants lacking KDR affinity; however, FLT-1 deficient mutants induced normal proliferation. These results suggest dual sets of determinants in the VEGF dimer that cross-link cell surface receptors, triggering endothelial cell growth and angiogenesis. Furthermore, this mutational analysis implicates KDR, but not FLT-1, in VEGF induction of endothelial cell proliferation.

Amino Acid Sequence↗
PubMed1996

A controlled trial of verapamil in patients after acute myocardial infarction: results of the calcium antagonist reinfarction Italian study (CRIS)

A multicenter, double-blind, randomized, placebo-controlled trial was conducted to assess the effects of verapamil on total mortality, cardiac mortality, reinfarction, and angina after an acute myocardial infarction. All patients, aged 30 to 75 years, consecutively admitted for acute myocardial infarction between 1985 and 1987 to the participating centers, and without contraindications to verapamil or history of severe heart failure were enrolled. Seven to 21 days (mean 13.8) after myocardial infarction, 531 patients were randomized to verapamil retard 360 mg/day, and 542 patients to placebo. At baseline, the 2 groups of patients had similar characteristics. Mean age was 55.5 years and 91% were men. During a mean follow-up of 23.5 months, 5.5% of the patients died. No differences between verapamil and placebo were observed in total mortality (n = 30 and 29, respectively) and cardiac death (n = 21 and 22, respectively). The verapamil group had nonsignificant lower reinfarction rates (n = 39 vs 49). The number of patients developing angina was significantly less in the verapamil group (n = 100 vs 132, RR = 0.8, 95% confidence interval 0.5 to 0.9). There were no differences in discontinuation of therapy caused by adverse reactions. This trial showed no effect of verapamil on mortality. The lower reinfarction rates found in the verapamil group are in agreement with the results of other studies.

Adult↗
PubMed1996

Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate.

OBJECTIVE: To determine if the angiogenic peptide vascular endothelial growth factor (VEGF) is required for retinal ischemia-associated iris neovascularization in a nonhuman primate. METHODS: Laser retinal vein occlusion was used to produce retinal ischemia in 16 eyes of eight animals (Macaca fascicularis). Eyes were randomized to treatment every other day with intravitreal injections of either a neutralizing anti-VEGF monoclonal antibody or a control monoclonal antibody of the same isotype. Serial iris fluorescein angiograms were assessed using a standardized grading system and masked readers. Retinal VEGF and placental growth factor expression were assessed by Northern blotting. The specificity of the antibodies was determined in capillary endothelial cell proliferation assays prior to intravitreal injection. RESULTS: Zero of eight eyes receiving the neutralizing anti-VEGF antibodies developed iris neovascularization. Five of eight control antibody-treated eyes developed iris neovascularization. The difference was statistically significant (P = .03). Intravitreal antibody injection did not impair the ability of the ischemic retina to increase VEGF messenger RNA expression. The anti-VEGF antibodies specifically inhibited VEGF-driven capillary endothelial cell proliferation in vitro. CONCLUSION: These data demonstrate that VEGF is required for iris neovascularization in an adult nonhuman primate eye. The inhibition of VEGF is a new potential therapeutic strategy for the treatment of ocular neovascularization.

Animals↗
PubMed1996

Vascular endothelial growth factor is sufficient to produce iris neovascularization and neovascular glaucoma in a nonhuman primate.

OBJECTIVE: To determine whether the angiogenic peptide vascular endothelial growth factor (VEGF) is sufficient to produce iris neovascularization in a nonhuman primate (Macaca fascicularis). METHODS: Eight eyes of 4 animals were studied. The 165-amino acid isoform of human recombinant VEGF (VEGF165) was injected into the vitreous of 5 cynomolgus monkey eyes (doses ranging from 0.25-2.5 micrograms per injection). Equal amounts of inactivated human recombinant VEGF (2 eyes) or vehicle (1 eye) were injected into contralateral control eyes. Eyes were assessed by slitlamp biomicroscopy, tonometry, iris color photography, fluorescein angiography, histopathologic examination, and immunostaining with antibodies against proliferating cell nuclear antigen. RESULTS: All 5 bioactive VEGF-injected eyes developed neovascularization with dilated and tortuous iris vessels that leaked fluorescein. None of the 3 control eyes exhibited any iris vascular changes. Inflammation was absent in both treatment groups. A dose response to VEGF was observed in the single animal that received 2.5 micrograms and 0.25 microgram in the right and left eyes, respectively. Iris vessel endothelial cells were positive for proliferating cell nuclear antigen in the bioactive VEGF-injected eyes only. Injections of 1.25 micrograms of VEGF every 3 days during a 30-day period produced advanced iris neovascularization, ectropion uvea, and neovascular glaucoma. CONCLUSIONS: Intravitreal injections of recombinant human VEGF165 in amounts comparable with those measured in eyes with active neovascularization are sufficient to produce noninflammatory iris neovascularization in a nonhuman primate. Prolonged exposure to VEGF165 can produce ectropion uveae and neovascular glaucoma.

Animals↗
PubMed1996

Crystallization of the receptor binding domain of vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor with a unique specificity for vascular endothelial cells. In addition to its role in vasculogenesis and embryonic angiogenesis, VEGF is implicated in pathologic neovascularization associated with tumors and diabetic retinopathy. Four different constructs of a short variant of VEGF sufficient for receptor binding were overexpressed in Escherichia coli, refolded, purified, and crystallized in five different space groups. In order to facilitate the production of heavy atom derivatives, single cysteine mutants were designed based on the crystal structure of platelet-derived growth factor. A construct consisting of residues 8 to 109 was crystallized in space group P2(1), with cell parameters a = 55.6 A, b = 60.4 A, c = 77.7 A, beta = 90.0 degrees, and four monomers in the asymmetric unit. Native and derivative data were collected for two of the cysteine mutants as well as for wild-type VEGF.

Binding Sites↗
PubMed1996

Hepatocyte growth factor. A cytokine mediating endothelial migration in inflammatory arthritis.

OBJECTIVE: Angiogenesis is an integral component of the vasculoproliferative phase of rheumatoid arthritis (RA). Recently, a heparin-binding cytokine termed hepatocyte growth factor (HGF), or scatter factor (due to its ability to disperse cohesive epithelial colonies), was described. We conducted this study to investigate the hypothesis that this cytokine was present in the milieu of the inflamed joint, and that it contributed to the chemotaxis of endothelial cells in the synovial tissue. METHODS: We examined synovial fluid, synovial tissue, and peripheral blood from 91 patients with RA and other arthritides. We used 83 total samples in an enzyme-linked immunosorbent assay to quantitate the HGF in synovial fluids and peripheral blood. To determine whether the HGF was biologically active, an epithelial scatter factor assay was performed. Immunohistochemical analysis was used to determine localization in synovial tissues. To define a function for synovial HGF, we preincubated rheumatoid synovial fluids with neutralizing anti-HGF and measured the ability of these synovial fluids to induce endothelial chemotaxis. RESULTS: Synovial fluid from patients with RA contained a mean +/- SEM HGF concentration of 2.0 +/- 0.3 ng/ml, while synovial fluid from patients with other arthritides (including inflammatory arthritis) contained 2.4 +/- 0.7 ng/ml HGF. Osteoarthritis (OA) patient samples contained the smallest quantities of synovial fluid HGF at 0.9 +/- 0.1 ng/ml. RA synovial fluid contained significantly more HGF than did RA peripheral blood (1.1 +/- 0.2 ng/ml) (P < 0.05). Rheumatoid synovial fluids induced more scattering of cells than did OA synovial fluids, suggesting a role for this cytokine in rheumatoid joint destruction. Interleukin-1 beta induced expression of rheumatoid synovial tissue fibroblast antigenic HGF and scatter factor activity. Immunohistochemically, HGF, as well as the HGF receptor (the met gene product), localized to significantly more rheumatoid synovial tissue lining cells than normal lining cells (P < 0.05). Both HGF and its receptor immunolocalized to subsynovial macrophages as well. Levels of synovial tissue immunoreactive HGF correlated positively with the number of synovial tissue blood vessels. Anti-HGF neutralized a mean of 24% of the chemotactic activity for endothelial cells found in 10 rheumatoid synovial fluid samples. CONCLUSION: These results indicate that synovial HGF may contribute to the vasculoproliferative phase of inflammatory arthritides such as RA, by inducing HGF-mediated synovial neovascularization. These findings point to a newly described role for HGF in the fibroproliferative phase of RA-associated synovitis.

Arthritis, Rheumatoid↗
PubMed1996

Preconditioning does not prevent postischemic dysfunction in aging heart.

OBJECTIVES: This study was performed to investigate the effect of single or multiple brief periods of ischemia and the administration of exogenous norepinephrine before a more prolonged ischemic period and after reperfusion in adult and senescent isolated and perfused rat hearts. BACKGROUND: The mortality rate for coronary artery disease is greater in the elderly. Ischemic preconditioning has been proposed as an endogenous form of protection against ischemia-reperfusion injury. However, the role of preconditioning in aging heart is unknown. METHODS: We compared the protective effect of preconditioning transient ischemic and norepinephrine stimuli against 20 min of global normothermic ischemia and 40 min of reperfusion in isolated perfused hearts of adult (6 months old) and senescent (24 months old) rats. Norepinephrine release in coronary effluent was determined by high performance liquid chromatography. RESULTS: Final recovery of percent developed pressure was improved after single preconditioning transient ischemic and norepinephrine stimuli in adult hearts (87.7 +/- 9% and 82.3 +/- 8.7%) versus unconditioned control hearts (50.6 +/- 4.8%, p < 0.01 [mean +/-SD]). The effect of preconditioning on developed pressure recovery was not present in senescent hearts after transient ischemic stimulus (39.8 +/- 4.9% vs. 41.6 +/- 5.8%, p = NS) but was present after norepinephrine stimulus (74.3 +/- 10.5, p < 0.01). Norepinephrine release significantly increased after preconditioning transient ischemic stimulus in adult but not in senescent hearts (p < 0.01 vs. adult). Transient ischemic- and norepinephrine-induced preconditioning was blocked by alpha-adrenergic receptor antagonists in both adult and senescent hearts. Multiple transient ischemic stimuli were able to reduce postischemic dysfunction in adult but not in senescent hearts. CONCLUSIONS: Preconditioning transient ischemic stimulus significantly reduces postischemic dysfunction in adult but not in senescent hearts, whereas exogenous norepinephrine is able to mimic preconditioning in both adult and senescent hearts. Ischemic preconditioning induces an increase in norepinephrine release in adult but not in senescent hearts. Preconditioning induced by transient ischemic stimulus and norepinephrine was abolished by alpha-adrenergic receptor blockade in both adult and senescent hearts. Thus, our data demonstrate that preconditioning is absent in aging heart and is probably related to the reduction of norepinephrine release and alpha-adrenergic receptor stimulation in response to ischemic preconditioning.

Aging↗
PubMed1996

Intravitreous injections of vascular endothelial growth factor produce retinal ischemia and microangiopathy in an adult primate.

PURPOSE: The purpose of the study is to determine the effect of exogenous vascular endothelial growth factor (VEGF) on the primate retina and its vasculature. METHODS: Ten eyes of five animals were studied. Physiologically relevant amounts of the 165 amino acid isoform of human recombinant VEGF were injected into the vitreous of six healthy cynomolgus monkey eyes. Inactivated human recombinant VEGF or vehicle was injected into four contralateral control subject eyes. Eyes were assessed by slit-lamp biomicroscopy, tonometry, fundus color photography, fundus fluorescein angiography, light microscopy, and immunostaining with antibodies against proliferating cell nuclear antigen and factor VIII antigen. RESULTS: All six bioactive VEGF-injected eyes developed dilated, tortuous retinal vessels that leaked fluorescein. Eyes receiving multiple injections of VEGF developed progressively dilated and tortuous vessels, venous beading, edema, microaneurysms, intraretinal hemorrhages and capillary closure with ischemia. The severity of the retinopathy correlated with the number of VEGF injections. None of the four control eyes exhibited any abnormal retinal vascular changes. The endothelial cells of retinal blood vessels were proliferating cell nuclear antigen positive only in the bioactive VEGF-injected eyes. CONCLUSION: Vascular endothelial growth factor is sufficient to produce many of the vascular abnormalities common to diabetic retinopathy and other ischemic retinopathies, such as hemorrhage, edema, venous beading, capillary occlusion with ischemia, microaneurysm formation, and intraretinal vascular proliferation.

Animals↗
PubMed1996

The role of aging on the control of contractile force by Na(+)-Ca2+ exchange in rat papillary muscle.

BACKGROUND: Sarcolemmal Na(+)-Ca2+ exchange system is believed to be fundamental to the control of cardiac contractility. However, the relation between Na(+)-Ca2+ exchange and the control of contractile force has not been studied in senescent myocardium. METHODS: The role of Na(+)-Ca2+ exchange in the regulation of the cardiac muscle's contractile force was studied in adult and senescent papillary muscles by simultaneously measuring intracellular sodium activity (aNai), action potential, and contractile force while varying extracellular concentration of sodium and/or calcium. RESULTS: Reduction of [Na+]o decreased aNai from 8.0 +/- 1.8 to 4.1 +/- 0.8 in adult (-3.9 mM) and from 8.7 +/- 1.9 to 4.7 +/- 0.9 in senescent (-4.0 mM) papillary muscles, while developed tension (DT) increased by 80.2% in adult and by 135.6% in senescent papillary muscles (p < .01 vs adult). During low [Ca2+]o and high [Na+]o, aNai and DT modifications were similar both in adult and senescent papillary muscles. During high [Ca2+]o, aNai decreased to a similar extent in both adult and senescent papillary muscles, while DT increased by 37.8% in adult and by 67.8% in senescent (p < .05 vs adult). Simultaneous reduction of [Na+]o and [Ca2+]o decreased aNai from 8.1 +/- 1.2 to 6.8 +/- 1.1 mM in adult (-1.3 mM), and from 8.4 +/- 1.0 to 7.2 +/- 1.0 mM in senescent (-1.2 mM) papillary muscles while DT decreased by 22.1% in adult and by only 12.0% in senescent (p < .01 vs adult) papillary muscles. Simultaneous increase of [Na+]o and [Ca2+]o similarly increased aNai in both adult senescent papillary muscles, but decreased DT by 28.5% in adult and by 11.7% in senescent (p < .01 vs adult). After [Na+]o modifications, the equilibration time for the ratio of external and internal sodium ion activities was slowed in senescent papillary muscles (i.e., in low [Na+]o solution the equilibration time was 4.6 +/- 0.9 min in adult and 6.3 +/- 1.2 min in senescent papillary muscles, p < .001). CONCLUSIONS: Similar changes of aNai during transmembrane Na+ and Ca2+ gradients modifications associated to changes in contractile force seem to demonstrate that Na(+)-Ca2+ exchange is probably not modified by the aging process. However, the slow equilibration time for the ratio of Na+ activities might reflect an age-related reduction of the Na(+)-K+ pump activity.

Action Potentials↗
PubMed1996

Vascular endothelial growth factor augments muscle blood flow and function in a rabbit model of chronic hindlimb ischemia.

Animal studies have shown that angiogenic factors can increase vascularity and improve blood pressure (BP) in an ischemic limb. Whether changes in these parameters are indicators of significant improvement in muscle function has not been demonstrated. In a rabbit model of hind limb ischemia, we measured blood flow in the extensor digitorum longus muscle (EDL) both at rest and during electrical stimulation. Ablation of the femoral artery caused significant reductions in resting and stimulated EDL blood flow. The chronic reduction in perfusion caused impairment of muscle function (p < 0.01). At 28 days after a single administration of vascular endothelial growth factor (VEGF), stimulated muscle blood flow (3 mg/kg intravenously, i.v.) and muscle function [1 mg intrarterially (i.a.) or 3 mg/kg i.v.] were significantly improved as compared with that of vehicle-treated controls. Simultaneous measurement of the hemodynamic responses in the contralateral limb and in the kidneys confirmed that the effects of VEGF were confined to the ischemic limb. The data agree with findings that angiogenic factors increase perfusion through angiogenesis. We hypothesized that neovascularization allows work-associated muscle hyperemia, resulting in a significant improvement in muscle function. Similar clinical improvements in muscle function would signify a substantial advance in the treatment of peripheral vascular disease.

Angiogenesis Inducing Agents↗
PubMed1996

Effects of vascular endothelial growth factor on hemodynamics and cardiac performance.

Vascular endothelial growth factor (VEGF), a major regulator of angiogenesis, has therapeutic benefit in animal models of coronary or limb ischemia. However, the hemodynamic effects of VEGF have not been investigated. We examined the effects of VEGF on hemodynamics and cardiac performance. Mean arterial pressure (MAP), heart rate (HR), cardiac output, stroke volume, left ventricular (LV) dP/dt, and hematocrit were measured before and after intravenous injection of VEGF in conscious, instrumented rats. VEGF caused a dose-dependent reduction in MAP and an associated increase in HR. VEGF (250 micrograms/kg) significantly decreased cardiac output and stroke volume without affecting the inotropic state of the left ventricle, as determined by dP/dt. VEGF significantly increased hematocrit. Furthermore, VEGF did not affect contractility or HR in the isolated rat heart in vitro. The data suggest that the VEGF-induced decrease in cardiac output is due to reduced stroke volume, which may be caused by a decrease in venous return rather than a direct effect on myocardial contractility. In addition, pretreatment with N omega-nitro-L-arginine methyl-ester (L-NAME), a nitric oxide (NO) synthase inhibitor, significantly attenuated the depressor and tachycardic responses to VEGF, suggesting that VEGF-induced hypotension may be mediated by NO.

Animals↗
PubMed1996

Vascular endothelial growth factor, a specific regulator of angiogenesis.

Vascular endothelial growth factor is a diffusible endothelial cell-specific mitogen and angiogenic factor that can also increase vascular permeability. The vascular endothelial growth factor receptors are specifically expressed on the cell surface of vascular endothelial cells. Recent studies point to vascular endothelial growth factor as a major regulator of physiological angiogenesis, such as developmental and reproductive angiogenesis. In addition vascular endothelial growth factor appears to be a crucial mediator of blood vessel growth associated with tumors and proliferative retinopathies. Antivascular endothelial growth factor antibodies have the ability to suppress the growth of a variety of tumor cell lines in nude mice and can also inhibit angiogenesis in animal models of intra-ocular neovascularization. Furthermore vascular endothelial growth factor administration promotes collateral vessel growth and results in functional improvement in animal models of coronary or limb ischemia.

Animals↗
PubMed1996

Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis.

The human endometrium undergoes a complex process of vascular and glandular proliferation, differentiation, and regeneration with each menstrual cycle in preparation for implantation. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic protein that appears to play an important role in both physiological and pathological neovascularization. To investigate whether VEGF may regulate human endometrial angiogenesis, we examined VEGF messenger ribonucleic acid (mRNA) and protein throughout the menstrual cycle and studied the regulation of VEGF by reproductive steroids in isolated human endometrial cells. By ribonuclease protection analysis, VEGF mRNA increased relative to early proliferative phase expression by 1.6-,2.0-, and 3.6-fold in midproliferative, late proliferative, and secretory endometrium, respectively. In histological sections, VEGF mRNA and protein were localized focally in glandular epithelial cells and more diffusely in surrounding stroma, with greatest VEGF expression in secretory endometrium. Consistent with these in vivo results, the treatment of isolated human endometrial cells with estradiol (E2), medroxyprogesterone acetate (MPA), or E2 plus MPA significantly increased VEGF mRNA expression over the control value by 3.1-, 2.8-, and 4.7-fold, respectively. The VEGF response to E2 was rapid, with steady state levels of VEGF mRNA reaching 85% maximum 1 h after the addition of steroid. E2 also caused a 46% increase in secreted VEGF protein, and the combination of E2 and MPA caused an 18% increase. VEGF expression in endometriosis, an angiogenesis-dependent, estrogen-sensitive disease was similar to that seen in eutopic endometrium. Peritoneal fluid concentrations of VEGF were significantly higher in women with moderate to severe endometriosis than in women with minimal to mild endometriosis or no disease. VEGF, therefore, may be important in both physiological and pathological angiogenesis of human endometrium, as it is an estrogen-responsive angiogenic factor that varies throughout the menstrual cycle and is elevated in women with endometriosis.

Cells, Cultured↗