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Biomedical subjects

H J Granger

Publications and source records attributed to H J Granger.

At least 37 records · Page 2Linked to original sources
PubMed1997

Distinct capillary density and progression promoted by vascular endothelial growth factor-A homodimers and heterodimers.

The aim of this study was to characterize the capillary density, progression and persistence of new capillaries induced by different isoforms of vascular endothelial growth factor (VEGF)-A. They were produced and purified using the same protocol and assessed in the same experimental model, the rabbit cornea assay. Monogenic homodimers for VEGF121 and VEGF165 together with the heterodimer VEGF121/165 were tested as slow release polymer pellets implanted into the avascular rabbit cornea and examined up to 18 days post-implantation. The implants consistently stimulated angiogenesis in the absence of inflammation. The VEGF121 isoform produced the strongest increase of new capillary vessels which rapidly and persistently progressed into the corneal stroma. VEGF165 promoted the growth of a smaller number of capillaries which ten-ded to regress over time. Heterodimers of VEGF121/165 produced intermediate in vivo activities between the two homodimers. In vitro endothelial cell proliferation, mobilization and adhesion were promoted by all VEGF isoforms under serum-free or serum-reduced conditions with the same order of potency. Anti-soluble KDR (sKDR) antibody completely inhibited the effects of all the isoforms. These results indicate that monogenic homodimer preparations of VEGF121 and VEGF165 can display distinct biological effects which are functionally retained after the heterodimeric assembly of VEGF121 and VEGF165. The observed different biological behavior of the VEGF isoforms reveals the possibility that in vivo the assembly of dimers derived from splicing of a single gene may yield molecules with either different matrix or receptor interaction, stability or diffusion rate according to specific needs.

Journal Article↗
PubMed1997

Nitric oxide promotes proliferation and plasminogen activator production by coronary venular endothelium through endogenous bFGF.

We reported previously that NO is responsible for the angiogenesis produced by endothelium-dependent vasodilating peptides. To investigate the mechanisms by which NO controls angiogenesis, NO was assessed for the ability to affect cell proliferation and upregulation of urokinase-type plasminogen activator (uPA) induced by basic fibroblast growth factor (bFGF) when added exogenously to or when produced endogenously by coronary venular endothelial cells (CVECs). The treatment of the cells with the NO donor sodium nitroprusside (NaNp) induced uPA upregulation and cell proliferation, which were prevented by anti-bFGF antibodies. Similarly, the NO-dependent mitogenic activity of the vasodilating peptide substance P (SP) was blocked by anti-bFGF antibodies, thus implicating endogenous bFGF in the NO-induced response. NaNp and SP induced bFGF expression as measured by Western blot analysis of CVEC extracts and by differential reverse transcriptase-polymerase chain reaction of bFGF mRNA. SP-induced upregulation of bFGF was prevented by the NO synthase inhibitor N omega-monomethyl-L-arginine. We conclude that NO promotes cell proliferation and uPA upregulation in CVECs by inducing endogenous bFGF and that this pathway mediates the angiogenetic response to the vasoactive neuropeptide SP. This signaling paradigm may provide an important link between shear rate, NO, bFGF, and coronary angiogenesis.

Cell Division↗
PubMed1997

Placenta growth factor-1 is chemotactic, mitogenic, and angiogenic.

The placental-derived growth factor (PIGF) is a dimeric glycoprotein showing a high degree of sequence similarity to the vascular endothelial growth factor. Alternative splicing of the PIGF primary transcript gives rise to two forms, named PIGF-1 and PIGF-2, which differ only in the insertion of a highly basic 21-amino acid stretch at the carboxyl end. The presence of the PIGF mRNA in thyroid, placenta, lung, and goiter has indicated the tissues where this factor functions. However, the role of PIGF in vascular development has not yet been clearly established. In the present study, we described the purification of PIGF-1 from overexpressing eukaryotic cells and then measured the angiogenic activity of the purified PIGF-1 in vivo in the rabbit cornea and the chick chorioallantoic membrane assays. In both in vivo assays, PIGF-1 induced a strong neovascularization process that was blocked by affinity-purified anti-PIGF-1 antibody. In the avascular cornea, PIGF-1 induced angiogenesis in a dose-dependent manner and seemed to be at least as effective (if not more effective) than vascular endothelial growth factor and basic fibroblast growth factor under the same conditions and at the same concentration. PIGF-1 was shown to induce cell growth and migration of endothelial cells from bovine coronary postcapillary venules and from human umbilical veins. In these two in vitro assays, PIGF-1 seemed to have a comparable effect to that of vascular endothelial growth factor and basic fibroblast growth factor on the cultured microvascular endothelium (eg, capillary venule endothelial cells). In summary, this is the first study to demonstrate that PIGF-1 can induce angiogenesis in vivo and stimulate the migration and proliferation of endothelial cells in vitro.

Allantois↗
PubMed1996

The effect of linomide on the migration and the proliferation of capillary endothelial cells elicited by vascular endothelial growth factor.

In order to assess the mechanism of action of the quinoline-3-carboxyamide linomide as an antiangiogenic drug, the effect of linomide was studied in vitro on postcapillary endothelial cells exposed to vascular endothelial growth factor (VEGF). Linomide did not block the spontaneous replication of endothelial cells, but significantly suppressed endothelial cell growth and migration elicited by VEGF. It is concluded that linomide appears to be an effective tool to inhibit VEGF-dependent angiogenesis.

Capillaries↗
PubMed1996

Nitric oxide mediates mitogenic effect of VEGF on coronary venular endothelium.

Vascular endothelial growth factor (VEGF) is a secreted protein that is a specific growth factor for endothelial cells. We have recently demonstrated that nitric oxide (NO) donors and vasoactive peptides promoting NO-mediated vasorelaxation induce angiogenesis in vivo as well as endothelial cell growth and motility in vitro; in contrast, inhibitors of NO synthase suppress angiogenesis. In this study we investigated the role of NO in mediating the mitogenic effect of VEGF on cultured microvascular endothelium isolated from coronary postcapillary venules. VEGF induced a dose-dependent increase in cell proliferation and DNA synthesis. The role of NO was determined by monitoring proliferation or guanosine 3',5'-cyclic monophosphate (cGMP) levels in the presence and absence of NO synthase blockers. The proliferative effect evoked by VEGF was reduced by pretreatment of the cells with NO synthase inhibitors. Exposure of the cells to VEGF induced a significant increment in cGMP levels. This effect was potentiated by superoxide dismutase addition and was abolished by NO synthase inhibitors. VEGF stimulates proliferation of postcapillary endothelial cells through the production of NO and cGMP accumulation.

Cell Line↗
PubMed1996

Acidic and basic FGFs dilate arterioles of skeletal muscle through a NO-dependent mechanism.

Fibroblast growth factors (FGFs) have been known to be potent stimulators of vascular endothelial cell proliferation and angiogenesis. Recent experimental evidence indicates that basic FGF (bFGF) is also involved in modulation of arterial pressure. In this study, we investigated the effects of acidic FGF (aFGF) and bFGF on muscle microcirculation using isolated arterioles and intact cremaster muscles of the at. In isolated microvessels, aFGF and bFGF (10(-12)-10(-8) M) significantly increased arteriolar diameter in a dose-dependent and time-dependent manner. This effect was abolished during inhibition of nitric oxide synthesis by NG-monomethyl-L-arginine (L-NMMA, 10(-4) M) but was not affected by indomethacin (10(-4) M), an inhibitor of the cyclooxygenase pathway of arachidonic acid metabolism. The vasodilation induced by FGFs was not observed in endothelium-denuded vessels. Furthermore, we studied microvascular hemodynamics in response to the growth factors in the cremaster muscle using intravital microscopy. Both aFGF and bFGF dilated arterioles of the intact cremaster muscle in a pattern similar to that observed in the isolated arterioles. At a concentration of 10(-10) M, aFGF caused a 19% increase in vessel diameter and 56% increase in blood flow. Administration of L-NMMA blocked by FGF-induced vasodilation and hyperemia. These results suggest that FGFs modulate blood flow in the skeletal muscle by acting on the endothelium of arterioles. The signaling mechanism of FGF-induced vasodilation involves the synthesis of nitric oxide by arteriolar endothelium.

Animals↗
PubMed1996

VEGF induces NO-dependent hyperpermeability in coronary venules.

The purpose of this study was to investigate the direct effect of vascular endothelial growth factor (VEGF) on microvascular permeability and its signaling mechanisms. The apparent permeability coefficient to albumin was measured in isolated coronary venules. Topical application of VEGF dose-dependently and transiently increased albumin permeability by two- to threefold. Inhibition of nitric oxide (NO) synthesis with NG-monomethyl-L-arginine abolished VEGF-induced venular hyperpermeability. Furthermore, because NO exerts vasoactive effects through stimulation of guanylate cyclase (GC) and the subsequent production of guanosine 3',5'-cyclic monophosphate (cGMP), we examined the role of GC and cGMP-dependent protein kinase (PKG) in the mediation of VEGF's action. The permeability response to VEGF was measured in the presence of the selective GC inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one and the specific PKG inhibitor KT-5823. Both inhibitors reduced basal permeability and prevented the hyperpermeability response to VEGF. Therefore, we suggest that VEGF modulates microvascular permeability via a signaling cascade involving NO synthesis, GC stimulation, and PKG activation.

Alkaloids↗
PubMed1995

Interaction of neutrophils and endothelium in isolated coronary venules and arterioles.

This study reports measurements of porcine neutrophil dynamics in isolated microvessels. Porcine coronary venules and arterioles were isolated, cannulated, and perfused with fluorescently labeled neutrophils at a series of flow velocities. In venules (62.50 +/- 5.41 microns diam) under control conditions, rolling neutrophils were often observed at intraluminal flow velocities ranging from 600 to 6,000 microns/s, and the rolling fraction varied inversely as a function of flow velocity. There was no significant adherence under the control conditions at any of the various flow velocities. Pretreatment of the neutrophils with human recombinant complement 5a (C5a, 10(-8) M) increased adherence at low flow velocities but did not alter the rolling fraction. In contrast to venules, rolling neutrophils were not observed in arterioles (58.80 +/- 5.6 microns diam). Furthermore, neutrophils that were pretreated with C5a did not adhere to the arteriolar endothelium even at low flow velocities. We suggest that 1) isolated microvessels perfused with fluorescently labeled neutrophils are suitable models for the study of the interaction between neutrophils and the microvascular endothelium, 2) shear force plays an important role in neutrophil rolling in coronary venules but is not the major factor that prevents neutrophil rolling and adherence in arterioles, and 3) C5a causes neutrophil adherence in venules but not in arterioles, indicating that different mechanisms underlie the interaction between neutrophils and endothelium in venules and arterioles.

Animals↗
PubMed1995

Oxidative injury of coronary venular endothelial cells depletes intracellular glutathione and induces HSP 70 mRNA.

Vascular endothelium is one of the first tissues exposed to reactive oxygen species produced during myocardial ischemia-reperfusion. Bovine coronary venular endothelial cells (CVEC) were evaluated for intracellular glutathione (GSH) levels and heat shock protein 70 (HSP 70) mRNA and protein during in vitro oxidative stress. CVEC were incubated with 0.01875 U/ml xanthine oxidase (XO) and 0.5 mM hypoxanthine (HX) for 30 min and then allowed to recover for 0, 1, 2, or 3 h. Relative GSH levels were determined by evaluation of monochlorobimane fluorescence. GSH fluorescence was significantly lower in CVEC treated with XO+HX for 30 min than in controls. GSH fluorescence was also decreased in heat-shocked CVEC. After oxidative stress, GSH levels were higher than in controls at 1 h, but by 2 or 3 h after treatment, GSH fluorescence fell below control values. HSP 70 mRNA was induced in CVEC by a 30-min treatment with XO+HX exposure. These data suggest that CVEC respond to oxidative stress by reducing intracellular GSH levels and inducing HSP 70 mRNA, although significant increases in HSP 70 protein were not detected at the time points tested.

Animals↗
PubMed1994

Nuclear accumulation of exogenous basic fibroblast growth factor in endothelial, fibroblast, and myoblast cell lines results in diverse biological responses.

During studies comparing 125I-bFGF internalization between endothelial cells and other cell types, we found, unexpectedly, internalization and nuclear translocation of exogenously added 125I-bFGF in two cell lines: Chinese hamster ovary cells (CHO) and rat L6 myoblasts. These cell lines were previously reported to be devoid of FGF receptors. Furthermore, CHO cells showed a weak mitogenic response to added bFGF, while L6 cells were mitogenically unresponsive. By comparison, coronary venular endothelial cells (CVEC), BALB/c 3T3 fibroblasts, and BHK-21 cells, demonstrated internalization and nuclear translocation of added 125I-bFGF, and mitogenic responsiveness to the growth factor. Insulin alone stimulated DNA synthesis in all cell types, yet augmented bFGF-dependent DNA synthesis only in CVEC, 3T3, and BHK. All five cell types expressed FGF receptors as assessed by covalent crosslinking with 125I-bFGF and immunoblotting with anti-FGF receptor antibodies. Differing rates of cytoplasmic and nuclear accumulation of 125I-bFGF and partial inhibition of internalization by pretreatment of CVEC with chlorate support a recent model that bFGF can internalize by two mechanisms. Insulin did not significantly affect 125I-bFGF internalization or metabolism in any cell type. bFGF treatment resulted in weak inhibition of RNA synthesis in L6 cells. bFGF appears firmly bound to the nuclear matrix as little nuclear-bound 125I-bFGF in CVEC is released by DNAse I or RNAse A digestion, while washes with 0.5 M NaCl result in partial release. Nuclear bFGF may thus be involved in regulation of nuclear events (e.g., gene transcription and/or DNA replication).

3T3 Cells↗
PubMed1994

Tyrosine kinase inhibitors impair fibroblast growth factor signaling in coronary endothelial cells.

We examined the effect of various tyrosine kinase inhibitors on basic fibroblast growth factor (bFGF)-induced cell signaling and DNA synthesis in coronary venular endothelial cells (CVEC). Two tyrosine kinase inhibitors, genistein and methyl 2,5-dihydroxycinnamate, showed reversible, dose-dependent inhibition of bFGF-stimulated DNA synthesis in CVEC with half-maximal inhibitory concentrations of 12 and 3 microM, respectively. Both compounds exhibited preferential inhibition of bFGF vs. serum-induced DNA synthesis. bFGF stimulated increased tyrosine phosphorylation of CVEC cellular proteins, including the FGF receptor, which were visible within 1 min of treatment. Concomitant with their effect on DNA synthesis, both compounds exhibited dose-dependent inhibition of tyrosine phosphorylation of intracellular substrates induced by bFGF. A 2-h pretreatment of quiescent CVEC with genistein blocked nuclear translocation but not cytoplasmic internalization of bFGF, whereas the same treatment with methyl 2,5-dihydroxycinnamate inhibited both processes. These results suggest that activation of bFGF receptor tyrosine kinase activity plays a role in nuclear translocation of bFGF and initiation of DNA synthesis in endothelial cells.

Animals↗
PubMed1994

Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P.

We evaluated the effects of nitric oxide (NO) generators and endogenous production of NO elicited by substance P (SP) in the angiogenesis process. Angiogenesis was monitored in the rabbit cornea in vivo and in vitro by measuring the growth and migration of endothelial cells isolated from coronary postcapillary venules. The angiogenesis promoted in the rabbit cornea by [Sar9]-SP-sulfone, a stable and selective agonist for the tachykinin NK1 receptor, and by prostaglandin E1 (PGE1), was potentiated by sodium nitroprusside (SNP). Conversely, the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME), given systemically, inhibited angiogenesis elicited by [Sar9]-SP-sulfone and by PGE1. Endothelial cells exposed to SNP exhibited an increase in thymidine incorporation and in total cell number. Exposure of the cells to NO generating drugs, such as SNP, isosorbide dinitrate, and glyceryl trinitrate, produced a dose-dependent increase in endothelial cell migration. Capillary endothelial cell proliferation and migration produced by SP were abolished by pretreatment with the NO synthase inhibitors N omega-mono-methyl-L-arginine (L-NMMA), N omega-nitro-L-arginine (L-NNA), and L-NAME. Exposure of the cells to SP activated the calcium-dependent NO synthase. Angiogenesis and endothelial cell growth and migration induced by basic fibroblast growth factor were not affected by NO synthase inhibitors. These data indicate that NO production induced by vasoactive agents, such as SP, functions as an autocrine regulator of the microvascular events necessary for neovascularization and mediates angiogenesis.

Amino Acid Oxidoreductases↗
PubMed1993

Substance P increases cyclic GMP levels on coronary postcapillary venular endothelial cells.

The vasodilating effect of substance P (SP) at the microvascular level is endothelium-dependent. In the present study we evaluated whether SP activates nitric oxide (NO) production by venular endothelial cell. We evaluated NO activation by measuring cyclic GMP levels in cultured endothelial cells isolated from coronary postcapillary venules of bovine origin (CVEC). Our results indicate that 5 min exposure of CVEC to 10 nM SP doubled basal cyclic GMP levels. Cell treatment with the NO synthase inhibitor L-NMMA reduced the basal levels of cyclic GMP and abolished the effect of SP but did not modify the increase in cyclic GMP in response to exogenous NO. These data indicate that a) microvascular endothelium responds in an autocrine fashion to NO with increased cyclic GMP levels, b) SP activates cyclic GMP pathway through NO production.

Animals↗
PubMed1993

Microvascular pressure and albumin extravasation after leukocyte activation in hamster cheek pouch.

The effects of leukotriene B4 (LTB4) and N-formyl-methionyl-leucylphenylalanine (FMLP) on transmicrovascular exchange of fluorescein isothiocyanate (FITC)-labeled serum albumin and dextran (molecular mass, 150,000 Da) were evaluated in hamster cheek pouch utilizing measurements of microvascular pressure and global efflux of the macromolecular markers into the tissue suffusate. Exposure of the suffused pouch to 1.0 microM LTB4 failed to alter microvascular pressure from its control level. The microvascular clearance of FITC-albumin rose to a peak value of 1.7 times the control value at a LTB4 concentration of 1.0 microM; the rate of albumin extravasation did not change from the normal level at a concentration of 0.1 microM. The extravasation rate of FITC-dextran was elevated with both doses of LTB4; the leukocyte chemotactic agent elicited 2.4- and 9.7-fold increments in peak clearance of dextran at concentrations of 0.1 and 1.0 microM, respectively. FMLP elicited a twofold increase in venular and terminal arteriolar pressures at a concentration of 1 microM; at the same concentration, the albumin clearance rose 3.5-fold. The results suggest that albumin transport remains near the normal level after LTB4 elicits massive migration of leukocytes across the venular membrane. By contrast, albumin transport is greatly accelerated following exposure to the chemotactic peptide FMLP; ultrastructural and physiological evidence suggests that the leakage of albumin is augmented by increases in microvascular permeability and filtration pressure. The results also reinforce previous ultrastructural observations from our laboratory indicating that diapedesis per se is not a cause of microvascular hyperpermeability in inflammatory reactions.

Animals↗
PubMed1993

Distribution, propagation, and coordination of contractile activity in lymphatics.

The propagation and coordination of lymphatic contractions were studied in the mesentery of the rat small intestine using in situ microscopic observation. Indexes of lymphatic diameter were simultaneously measured at two adjacent lymphangions in spontaneously contracting lymphatics (n = 51). Diameter index, contraction frequency, and the percentage of the intersegmental contractions that were propagated and coordinated (PP) were determined at both sites. The conduction velocity of the contractile activity and the percentage of the coordinated contractions that were propagated both antegrade to the direction of lymph flow and retrograde to the flow stream were determined. The results indicate that 1) 80-90% of the lymphatic contractions in the vessels we evaluated were propagated, 2) the wave of contractile activity propagated both centrally and peripherally, and 3) the conduction velocity of the contractile activity was approximately 4-8 mm/s. We tested the hypothesis that gap junctional communication is responsible for the coordination of the contractile event. To accomplish this, we used the gap junction blockers n-heptanol and oleic acid. PP was 90 +/- 4% under normal conditions and fell to a minimum value of 55 +/- 7% during the gap junction blockade. These results indicate that gap junctional communication played an important role in the propagation and coordination of contractions that occurred in spontaneously active lymphatics.

Alcohols↗
PubMed1993

Histamine increases venular permeability via a phospholipase C-NO synthase-guanylate cyclase cascade.

In this study, we hypothesized that histaminergic increases in venular permeability result from a cascade triggered by activation of phospholipase C (PLC), inducing the synthesis of nitric oxide (NO) and activating guanylate cyclase. The apparent permeability coefficient to albumin (Pa) was measured in isolated porcine coronary venules subjected to constant flow and hydrostatic and oncotic pressures. Histamine (2.5, 5, and 10 microM) transiently and progressively increased Pa. The PLC inhibitor 2-nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC; 100 microM) decreased baseline permeability and abolished the effect of histamine. The NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 10 microM) and the guanylate cyclase inhibitor 6-anilinoquinoline-5,8-quinone (LY 83583; 10 microM) also blocked the histamine-induced hyperpermeability. L-Arginine (3 mM) reversed the inhibition by L-NMMA. NG-monomethyl-D-arginine did not influence the effect of histamine. Furthermore, sodium nitroprusside (10 microM) augmented Pa by two- to threefold; this effect was blocked in the presence of LY 83583 but not altered in the presence of NCDC. The results suggest that histamine increases coronary venular permeability by a direct action on the venular endothelial cells through a PLC-NO synthase-guanylate cyclase-signaling cascade.

Amino Acid Oxidoreductases↗
PubMed1993

Permeability to albumin in isolated coronary venules.

This study reports measurements of albumin permeability in isolated coronary venules. The isolated microvessel technique allows the quantification of transmural exchange of macromolecules under tightly controlled physical and chemical conditions. Transvenular exchange of albumin was studied in isolated coronary venules during alterations in filtration rate caused by changes in intravascular pressure. The apparent permeability coefficient of albumin (Pa) at an intraluminal pressure of 11 cmH2O was 3.92 +/- 0.43 x 10(-6) cm/s. Elevating intraluminal pressure to 16 and 21 cmH2O increased Pa to 5.13 +/- 0.57 x 10(-6) and 6.78 +/- 0.66 x 10(-6) cm/s, respectively. Calculation of the true diffusive permeability coefficient of albumin (Pd) at zero filtration rate was 1.54 x 10(-6) cm/s. The product of hydraulic conductance (Lp) and (1 - sigma), where sigma is the solute reflection coefficient, was 3.25 x 10(-7) cm.s-1 x cmH2O-1. At a net filtration pressure of 4-5 cmH2O, diffusion accounts for > 60% of total albumin transport across the venular wall. Transmural albumin flux is very sensitive to filtration rate, rising 6.7% for each cmH2O elevation of net filtration pressure. At 11 cmH2O net filtration pressure, convection accounts for nearly 70% of net albumin extravasation from the venular lumen. We suggest that the isolated coronary venule is a suitable preparation for the study of solute exchange in the heart.

Animals↗