Xiaoyou Ying

Pressure is proinflammatory in lung venular capillaries

Endothelial responses may contribute importantly to the pathology of high vascular pressure. In lung venular capillaries, we determined endothelial [Ca(2+)](i) by the fura-2 ratioing method and fusion pore formation by quantifying the fluorescence of FM1-43. Pressure elevation increased endothelial [Ca(2+)](i). Concomitantly evoked exocytotic events were evident in a novel spatial-temporal pattern of fusion pore formation. Fusion pores formed predominantly at vascular branch points and colocalized with the expression of P-selectin. Blockade of mechanogated Ca(2+) channels inhibited these responses, identifying entry of external Ca(2+) as the critical triggering mechanism. These endothelial responses point to a proinflammatory effect of high vascular pressure that may be relevant in the pathogenesis of pressure-induced lung disease.

Ca2+ Waves in Lung Capillary Endothelium

Although cytosolic Ca2+ importantly regulates organ function, lung microvascular [Ca2+]i regulation remains poorly understood because of the lack of direct in situ quantification. In the present study, we report the first endothelial [Ca2+]i quantification by the fura 2 method in microscopically imaged venular capillaries of the isolated blood-perfused rat lung. Sequential images indicated the presence of intercellular Ca2+ waves that spontaneously originated from pacemaker endothelial cells and then spread for short distances along the capillary wall, inducing synchronous endothelial [Ca2+]i oscillations. Fast Fourier analyses of the oscillations revealed a dominant wave component with an amplitude of 37 nmol/L, frequency of 0.4 min-1, and velocity of 5 microns/s. The intracellular Ca2+ wave was unaffected by blood flow stoppage or by infusions of Ca(2+)-containing or Ca(2+)-free dextran. Inhibition of the wave by thapsigargin in Ca(2+)-free dextran and by the gap junction uncoupler, heptanol, indicated that it was generated by endosomal Ca2+ release in the pacemaker cell and was propagated by gap junctional communication. In the presence of histamine, enhancement of the wave accounted for a significant component of the coordinated [Ca2+]i increase in the capillary segment. No intercellular Ca2+ waves were evident in adjoining alveolar epithelial cells. Our findings indicate a novel mechanism of [Ca2+]i regulation in the lung capillary under both resting and stimulated conditions. Pacemaker-induced Ca2+ waves, generated intracellularly by unknown initiating mechanisms, communicated to adjoining cells to determine [Ca2+]i profiles in short interbranch segments of capillary walls.

Ligation of Endothelial αvβ3 Integrin Increases Capillary Hydraulic Conductivity of Rat Lung

Complement-mediated pulmonary edema results from increases in lung capillary hydraulic conductivity (Lp), possibly by receptor-mediated mechanisms. We considered the Lp effects of vitronectin and the vitronectin-containing complement complex SC5b-9, which ligate the integrin alpha v beta 3. Vitronectin, SC5b-9, and SC5b-9-enriched zymosan-activated serum all rapidly increased Lp, as determined by the split-drop technique in single lung capillaries of rat lung. The Lp increases were inhibited by a monospecific (LM609) and a polyclonal (R838) antibody against the alpha v beta 3 integrin but not by an irrelevant monoclonal antibody isotype matched with LM609, by a monoclonal antibody against the alpha v beta 5 integrin, or by preimmune rabbit serum. Vitronectin monomers failed to increase Lp. The tyrosine kinase blockers genistein and methyl 2,5-dihydroxycinnamate caused significant concentration-dependent inhibitions of Lp increases due to vitronectin and zymosan-activated serum. By contrast, the protein kinase C blocker calphostin C had no major effect. We conclude that (1) multivalent ligation of the luminally located alpha v beta 3 integrin of lung capillary endothelium increases transcapillary liquid flux, and (2) the dominant signal transduction pathway for this effect occurs through tyrosine kinase activation.

αvβ3 Integrin Induces Tyrosine Phosphorylation–Dependent Ca2+ Influx in Pulmonary Endothelial Cells

Abstract—The endothelial αvβ3 integrin occurs luminally, where its ligation by soluble agents may induce inflammatory signaling. We tested this hypothesis in bovine pulmonary artery endothelial cell monolayers with the use of vitronectin and cross-linking antibodies to ligate and aggregate the integrin. We quantified the endothelial cytosolic Ca2+ concentration ([Ca2+]i) according to the Fura 2 ratio imaging method in single cells of confluent monolayers. At baseline, endothelial [Ca2+]i levels remained steady at 86 nmol/L for >20 minutes. Cross-linking of the αvβ3 integrin through the sequential exposure of monolayers to anti-αvβ3 monoclonal antibody LM609 and secondary IgG resulted in a [Ca2+]i increase of 100% above baseline. This increase commenced in <0.5 minute, peaked in <2 minutes, and decayed to baseline in ≈5 minutes. Similar responses occurred after the addition of vitronectin (400 μg/mL). In contrast, external Ca2+ depletion blunted the cross-linking–induced [Ca2+]i increase by 60%, a response...

SIGNAL TRANSDUCTION BY AN INFLAMMATORY INTEGRIN IN LUNG ENDOTHELIAL CELLS.|[dagger]| 1467

The lung endothelial αvβ3 integrin which is luminally located in lung capillaries, binds vitronectin-containing inflammatory products to increase capillary permeability (Circ. Res. 1995; 77:651-659). To determine signalling mechanisms, we determined cytosolic calcium concentration([Ca2+]i) by the fura method, in microvascular endothelial cells of rat lung (RLMEC). Vitronectin (400 μg/ml) increased [Ca2+]i of fura 2-loaded RLMEC by 176±30% from baseline of 92±14 nM, in 1 min and remained elevated for 5 min (P<.05; N=14). Immunoprecipitation of phospholipase Cγ (PLCγ) from RLMEC monolayers exposed to vitronectin, followed by immunoblotting with anti-phosphotyrosine(Bhattacharya, S., J. Biol. Chem. 1995; 270: 16781-87) revealed enhanced protein tyrosine phosphorylation (PtyrP) on PLCγ. Since activated PLCγ may increase inositol (1,4,5) triphosphate (IP3), we quantified IP3 by receptor-binding assay. In RLMEC monolayers, vitronectin treatment increased IP3 32±5% in 5 min (P<.05). We conclude, the signalling pathway induced by ligation of the lung endothleial αvβ3, sequentially consists of tyrosine phosphorylation of PLCγ, hence probable activation of PLCγ, release of IP3, then IP3-mediated increase of [Ca2+]i. Subsequently, increase of capillary permeability may be attributable to activation of Ca2+ dependent processes (HL54157, HL36024).

CROSS-LINKING THE ENDOTHELIAL αvβ3 INTEGRIN CAUSES CALCIUM-INDEPENDENT ENHANCEMENT OF PROTEIN TYROSINE PHOSPHORYLATION.▴ 2288

Cross-linking the αvβ3 integrin in the presence of extracellular Ca2+, enhances protein tyrosine phosphorylation in lung endothelial cells (Bhattacharya, S. et al, J. Biol. Chem. 270: 16781-16787, 1995). To determine the Ca2+-dependence of this response, we loaded rat lung microvascular endothelial cells (RLMVEC) with Fura-2AM (5 μM, 20 min, 20°C), then incubated them with the anti-αvβ3 polyclonal antibody, R838 (20 μg/ml, 30 min, 4°C). To cross-link theαvβ3 integrin, we exposed the monolayers to a goat anti-rabbit secondary antibody (5 min, 37°C). Then we determined cytosolic Ca2+([Ca2+]i) by Fura-2 imaging and protein tyrosine phosphorylation by SDS-PAGE and immunoblotting of cell lysates. Cross-linking in Ca2+-HEPES increased [Ca2+]i and enhanced tyrosine phosphorylation. However, cross-linking either in Ca2+-free HEPES or following cell incubation with the membrane permeant calcium chelator, MAPTAM(bis-(2-Amino-5-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid tetraacetoxymethyl ester; 200 μM), blunted the increase of[Ca2+]i but not the enhanced tyrosine phosphorylation. We conclude first, that in RLMVEC, cross-linking the αvβ3 integrin increases [Ca2+]i because of external calcium entry; second, that the cross-linking induced enhanced tyrosine phosphorylation is[Ca2+]i independent (supported by R29 HL54157).