Endothelial Mitogenic Activity Research Articles

Evaluating VEGF-Induced Vascular Leakage Using the Miles Assay.

Before the endothelial mitogenic activity of the Vascular Endothelial Growth Factor A (VEGF) was described, VEGF had already been identified for its ability to induce vascular leakage. VEGF-induced vascular leakage has been most frequently studied in vivo using the Miles assay, a simple yet invaluable technique that has allowed researchers to unravel the molecular mechanisms underpinning vascular leakage both for VEGF and other permeability inducing agents. In this protocol, a mouse is intravenously injected with Evans Blue dye before VEGF is administered locally via intradermal injection. VEGF promotes vascular leak of serum proteins in the dermis, enabling Evans Blue-labeled albumin extravasation from the circulation and subsequent accumulation in the skin. As the volume of dye extravasation is proportional to the degree of vascular leak, it can be quantified as a proxy measurement of VEGF-induced vascular leakage.

Characterization of heparin-binding endothelial mitogen(s) produced by the ovine endometrium during early pregnancy.

To characterize mitogenic factors produced by ovine endometrium during early pregnancy, endometrial explant-conditioned media (ECM) were obtained from ewes on day 12, 18, 24, or 30 after mating. These ECM contained mitogenic activity for both endothelial and 3T3 cells across all days. The endothelial mitogenic activity was greatest on day 24, whereas mitogenic activity for 3T3 cells did not differ across days. By ultrafiltration, ion exchange, and heparin-affinity chromatography, the endothelial mitogenic activity was found to have a molecular mass greater than 100 kDa, to be anionic, and to be heparin binding, respectively. Three peaks of endothelial mitogenic activity were recovered from heparin-affinity chromatography. The major peak, H3, was mitogenic for endothelial but not for 3T3 cells. H3 was further purified, and the single peak of heparin-binding activity, designated H3b, represented a 681-fold purification of endothelial mitogenic activity from endometrial ECM. H3 and H3b were heat labile and trypsin sensitive, and their biological activity was heparin enhanced. The majority of the endothelial mitogenic activity was immunoneutralized by antibodies against acidic and basic FGF. Nevertheless, we were unable to detect bFGF in H3 or H3b by Western immunoblot analysis. Thus, in this study we have extended our previous observations and demonstrated that (i) during early pregnancy the ovine endometrium produces mitogenic activity for both endothelial and 3T3 cells, (ii) the endothelial mitogenic activity is greatest on day 24 after mating. which corresponds with the onset of endometrial vascular growth, and (iii) the major endothelial mitogen has a high affinity for heparin, and although it is immunologically related to FGF, it differs from known FGF in its apparent molecular size and biological activities.

Expression of basic fibroblast growth factor, endothelial mitogenic activity, and angiotensin II type-1 receptors in the ovine placenta during the third trimester of pregnancy.

To evaluate expression of basic fibroblast growth factor (FGF), endothelial mitogenic activity, and angiotensin II type-1 receptors (AT1r), as well as the role of angiotensin II (ANG II) in regulating basic FGF production/secretion, placentae were obtained from ewes on Days 110, 120, 130, and 142 of pregnancy and were separated into fetal cotyledonary (COT) and intercotyledonary (ICOT), as well as maternal caruncular (CAR) and intercaruncular (ICAR) components. Using immunohistochemistry, basic FGF and AT1r were found for the most part to be colocalized in all placental components, primarily in epithelium, stroma, endothelium, and vascular smooth muscle. Changes in basic FGF levels in placental explant-conditioned media were observed in fetal, but not maternal, components. In COT, basic FGF levels increased 2.4-fold (r2 = 0.48, p < 0.04) from Day 110 to 130 and then declined at term. In ICOT, basic FGF levels increased 6.4-fold (r2 = 0.33, p < 0.05) from Day 110 to 142. The rank order of averaged basic FGF levels was COT > CAR > ICOT = ICAR (p < 0.05). Endothelial mitogenic activity of conditioned media was observed in COT from Day 130 pregnant ewes (232.5 +/- 38.7% of control; p < 0.05) but not in other components, and it was neutralized by a basic FGF antibody. ANG II did not alter basic FGF levels in any placental component. Thus, throughout the third trimester, 1) basic FGF and AT1r are present in placentae, 2) both basic FGF levels and endothelial mitogenic activity in COT increase, 3) basic FGF levels are associated with endothelial mitogenic activity of COT, and 4) ANG II has no effect on production/secretion of basic FGF by the placenta.

Initial characterization of endothelial mitogens produced by bovine corpora lutea from the estrous cycle.

To further characterize mitogenic factor(s) present in luteal extracts or luteal explant conditioned media (LCM), bovine corpora lutea (CL) were homogenized or incubated in explant culture, respectively. After evaluation of luteal extracts and LCM by using an endothelial cell proliferation bioassay, mitogenic activity was characterized by immunoneutralization with antibodies against heparin-binding (fibroblast) growth factor (HBGF) 1 or 2. LCM also were subjected to ultrafiltration, as well as anion-exchange, cation-exchange, and heparin-affinity chromatography. The presence of HBGF-2 in LCM also was evaluated by using a dot immunoblot assay. Extracts of luteal tissues and LCM stimulated (P < 0.05) proliferation of endothelial cells in a dose-dependent manner. Mitogenic activity of luteal extracts and LCM was decreased (P < 0.05) by treatment with specific antibodies against HBGF-2 or HBGF-1. LCM also contained immunoreactive HBGF-2. The mitogenic activity bound to anion exchangers, phenyl-Sepharose, and heparin-agarose, but not to cation exchangers, indicating that endothelial mitogenic activity is anionic at neutral pH, has some hydrophobic characteristics, and belongs to the HBGF family of proteins. Following ultrafiltration, endothelial mitogenic activity was retained by membranes having a 30,000 or 100,000 molecular weight cutoff. In addition, LCM was resolved into four peaks of heparin-binding endothelial mitogenic activity, each with a different affinity for heparin. These data demonstrate that bovine CL contain and produce endothelial mitogens of large molecular size, which may be important regulators of luteal function. These endothelial mitogens are heparin-binding and anionic at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)

Capillary endothelial-cell mitogenic activity in experimental branch vein occlusion

Since the 1940s, the major hypothesis regarding the stimulus for neovascularization occurring in diabetic retinopathy and other "ischemic" retinopathies has involved the release of a soluble angiogenic factor from the ischemic retina. We sought to test this by measuring the endothelial mitogenic activity that could be extracted from an ischemic retina caused by branch vein occlusion in the cat. We found that the extractable endothelial-cell mitogenic activity from normal retinal areas was similar to that in ischemic areas at 1, 2, and 7 days after the occlusion. In the area of occluded veins, the oxygen partial pressure was low (7 +/- 7 mm Hg) compared with the normal value (23 +/- 8 mm Hg). The data did not show any increase in soluble mitogenic factor release from the ischemic retina versus normal retinal areas under the conditions of this experiment.