Michael A. Gimbrone

Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2

In the face of systemic risk factors, certain regions of the arterial vasculature remain relatively resistant to the development of atherosclerotic lesions. The biomechanically distinct environments in these arterial geometries exert a protective influence via certain key functions of the endothelial lining; however, the mechanisms underlying the coordinated regulation of specific mechano-activated transcriptional programs leading to distinct endothelial functional phenotypes have remained elusive. Here, we show that the transcription factor Kruppel-like factor 2 (KLF2) is selectively induced in endothelial cells exposed to a biomechanical stimulus characteristic of atheroprotected regions of the human carotid and that this flow-mediated increase in expression occurs via a MEK5/ERK5/MEF2 signaling pathway. Overexpression and silencing of KLF2 in the context of flow, combined with findings from genome-wide analyses of gene expression, demonstrate that the induction of KLF2 results in the orchestrated regulation of endothelial transcriptional programs controlling inflammation, thrombosis/hemostasis, vascular tone, and blood vessel development. Our data also indicate that KLF2 expression globally modulates IL-1beta-mediated endothelial activation. KLF2 therefore serves as a mechano-activated transcription factor important in the integration of multiple endothelial functions associated with regions of the arterial vasculature that are relatively resistant to atherogenesis.

Fine structure of vascular endothelium in culture

Human umbilical cord vein endothelial cells were harvested by collagenase perfusion, cultured in Medium 199 with 20% fetal calf serum, and subcultured for 19 passages over one year. By phase microscopy, cultured endothelial cells were small and polygonal and in successful cultures, formed a monolayer of closely packed cells. By electron microscopy, cultured endothelium retained fine structural characteristics of in vivo umbilical vein endothelium. Specific endothelial granules first described by Weibel and Palade (30), were found regularly in all passages. Microtubules (250 A), two types of filaments with diameters of 100 A and 60–70 A, respectively, pinocytotic vesicles, multivesicular bodies, and cellular junctions were distributed in a way that cultured endothelium could be distinguished by morphologic criteria from the most probable contaminants, smooth muscle cells, and fibroblasts.

Endothelial-dependent mechanisms of leukocyte adhesion in inflammation and atherosclerosis.

Adhesion of circulating leukocytes to the blood vessel wall is an essential component of acute and chronic inflammatory reactions, and various vascular disease processes, including vasculitis, allograft rejection, ischemia-reperfusion injury, and atherosclerosis. In particular, the focal adherence of blood monocytes at certain sites in the aortic tree, and their subsequent transmigration into the intima, appear to be consistent early events in the formation of foam cell-rich lesions during atherogenesis in humans and experimental animals. ‘ I 6 There is increasing evidence that vascular endothelial cells play an active role in these processes. Our research group has been especially interested in defining endothelial-dependent mechanisms of leukocyte adhesion, and exploring their relevance for human vascular pathophysiology. In this paper, we shall summarize our recent progress in characterizing inducible endothelial cell surface structures involved in leukocyte adhesion-endothelial-leukocyte adhesion molecules (“ELAMs”). We will also provide a brief summary of the pathophysiologic implications of leukocyte-endothelial interactions for the atherosclerotic disease process, and indicate promising future directions for research in this area.

Endothelial cell adenylate cyclase: Activation by catecholamines and prostaglandin I2☆

Abstract Following incubation of intact vascular endothelial cells with 1 mM 3-isobutyl-1-methylxanthine, and isoproterenol or PGI 2 , cyclic AMP levels increased 4- and 3-fold, respectively. Isoproterenol-stimulated adenylate cyclase activity of cell lysates was selectively inhibited by the β-adrenergic blocking agent propranolol. Catecholamines stimulated adenylate cyclase with the potency series: isoproterenol > epinephrine > norepinephrine. Prostaglandin did not stimulate adenylate cyclase activity in cell lysates, even in the presence of guanine nucleotides or following preincubation of the intact cells with prostaglandins prior to freeze-thaw lysis.

C‐C and C‐X‐C Chemokines Trigger Firm Adhesion of Monocytes to Vascular Endothelium under Flow Conditionsa

In summary, our findings indicate that specific chemokines that are elaborated by endothelial cells after cytokine or endotoxin activation can play an essential role in monocyte recruitment beyond their chemoattractant activities. We show that this action is to translate initial monocyte tethering into firm adhesion via rapid leukocyte integrin activation. The in vitro model presented here provides a sensitive system for investigating the modulating ability of chemokines and reveals an important biological effect that is not predicted by results in simpler in vitro assays, such as measurement of calcium transients or chemotaxis. The surprising finding that the C-X-C chemokine IL-8 can trigger monocyte firm adhesion to vascular endothelium suggests a potential role for this chemokine in monocyte recruitment and underscores the biological complexity of the chemokine family.

Vascular lipoxygenase activity: synthesis of 15-hydroxyeicosatetraenoic acid from arachidonic acid by blood vessels and cultured vascular endothelial cells

Although indirect pharmacologic evidence has suggested the presence of a lipoxygenase pathway of arachidonic acid (AA) metabolism in blood vessels, direct biochemical evidence has been difficult to demonstrate. We have investigated lipoxygenase metabolism in both fresh vessel preparations and cultured vascular cells from various sources and species. Lipoxygenase-derived [3H] HETE (composed of 12-HETE, 15-HETE and 5-HETE), which was abolished by ETYA but not by aspirin, was formed when [3H]AA was incubated with fresh sections of rat aorta. Lipoxygenase activity was lost following deendothelialization. A single peak of [3H] 15-HETE was produced by cultured bovine aortic and human umbilical vein endothelial cells (EC) in response to exogenous [3H]AA or from [3H]AA released by ionophore A23187 from endogenous EC membrane phospholipid pools. Cultured bovine, rabbit or rat aorta smooth muscle cells had no detectable 15-lipoxygenase activity. [14C] Linoleic acid was converted by EC to its 15-lipoxygenase metabolite, [14C] 13-hydroxyoctadecadienoic acid. These results indicate that blood vessels from different sources and species have a 15-lipoxygenase system, and this activity resides predominantly in the endothelial cells.

Angiotensin-converting enzyme activity in isolated brain microvessels

The localization of angiotensin-converting enzyme (kininase II; ACE) in bovine cerebral cortex was studied by mechanically isolating microvessels from surrounding brain parenchyma. ACE specific activity, as assayed by generation of L-histidyl-L-leucine from the synthetic substrate hippuryl-L-histidyl-L-leucine, was enriched approximately 30 times in microvessels compared to homogenates of intact cerebral cortical gray matter. The nonapeptide <Gluue5f8Trpue5f8Proue5f8Argue5f8Proue5f8Glnue5f8Ileue5f8Proue5f8Pro (BPF9a, SQ20,881), the orally active anti-hypertensive drug, 2-D-methyl-3-mercaptopropanoyl-L-proline (SQ14,225), and the vasoactive peptides bradykinin and angiotensin II inhibited this activity in a dose-dependent fashion. Brain microvessel ACE required chloride for optimal activity, was potentiated by cobalt nitrate, and was inhibited by the chelating agents EDTA and o-phenanthroline. Enzymatic generation of histidyl-leucine also was observed with the naturally occurring decapeptide substrate angiotensin I. In addition, microvessels obtained from bovine cerebellar cortex, hippocampus and corpus striatum, as well as from the cerebral cortex of Sprague-Dawley rats, were enriched in ACE activity. The presence of angiotensin-converting enzyme in brain microvessels suggests that cellular components of the blood-brain barrier may participate in the metabolism of peptide hormones such as angiotensin I and bradykinin within the central nervous system.

Angiotensin metabolism by cultured human vascular endothelial and smooth muscle cells.

Human vascular endothelial and smooth muscle cells (SMC) were selectively cultured from umbilical cord veins and examined for angiotensin-converting enzyme (CE, kininase II). Angiotensin-converting activity was observed as a single peak in gel filtrates of mechanically harvested endothelial cells (EC). The enzyme released histidylleucine from angiotensin I and phenylalanylarginine from bradykinin, required Cl− for maximal activity, was partially inhibited by ethylenediamine-tetraacetic acid and strongly inhibited by Pyr-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro (SQ20881, BPF9a). Its molecular weight, estimated by gel filtration, was 140,000; by sucrose density gradient centrifugation, 115,000. No CE was found in conditioned medium from either EC or SMC cultures, nor in sonicates of SMCs. Significant destruction of angiotensin II was not detected in EC sonicates at pH 7.0 or 7.4, but substantial degradation did occur at pH 5.8. The ability of extrapulmonary vascular endothelium to generate angiotensin II, a potent vasopressor, and to degrade bradykinin, a vasodilator, provides further evidence for an active metabolic role of endothelium in the metabolism of vasoactive peptides.