Alfred W.A. Hahn

Endothelin stimulated by angiotensin II augments contractility of spontaneously hypertensive rat resistance arteries.

In cultured endothelial cells, endothelin is produced after stimulation with angiotensin II. The effects of angiotensin II and endothelin-1 on vascular sensitivity to norepinephrine were studied in perfused rat mesenteric resistance arteries. Expression of endothelin messenger RNA (mRNA) was determined in endothelial cells obtained from the mesenteric circulation. Perfusion (5 hours) of the arteries with angiotensin II (10(-7) M) potentiated contractions in arteries with endothelium induced by norepinephrine in spontaneously hypertensive rats but not Wistar-Kyoto rats. The potentiation was inhibited by phosphoramidon and an endothelin antibody. Short-term stimulation (1 hour) with angiotensin II did not cause the potentiation. Stimulation with angiotensin I (10(-7) M; 5 hours) caused a potentiation prevented by captopril. In endothelial cells collected from the mesenteric arterial bed of spontaneously hypertensive rats, endothelin-specific mRNA was constitutively expressed, and the level of endothelin transcripts was increased by angiotensin II (10(-7) M). Threshold concentrations of exogenous endothelin-1 potentiated contractions induced by norepinephrine in arteries with and without endothelium of spontaneously hypertensive rats but not Wistar-Kyoto rats. Thus, angiotensin II stimulates the endothelial production of endothelin in situ and therapy potentiates contractions to norepinephrine in mesenteric resistance arteries of spontaneously hypertensive rats. This suggests that vascular endothelin production acts as an amplifier of the pressor effects of the renin-angiotensin system that may play an important role in hypertension.

Inducibleendothelin mRNA expression and peptide secretion in cultured human vascular smooth muscle cells

Abstract This study demonstrates the induction of endothelin (ET) mRNA expression and synthesis of functional ET -peptide in cultured human vascular smooth muscle cells (hVSMC). Compounds eliciting such responses in hVSMC include the vasoconstrictor hormones angiotensin II and arginine-vasopressin and the growth factors transforming growth factor s, platelet derived growth factor AA and epidermal growth factor. Induction of ET mRNA expression in hVSMC exhibited transient kinetics (peak at 3–5 hrs. and return to basal within 7 hrs.) which differed from the more sustained ET transcript induction observed for porcine endothelial cells. ET peptide (determined by both radioimmuno-and radioreceptor assays) produced by stimulated hVSMC attained levels (∼ 120–160 pg/10 6 cells/4 hrs.; concentration ∼ 3 × 10 −11 M) within the biologically effective concentration range of ET. Stimulated secretion of ET from hVSMC was abolished in the presence of the protein synthesis inhibitor cycloheximide. Sep-pak C 18 extracts of medium from stimulated hVSMC elicited a concentration-dependent phosphoinositide catabolic response in myo-[2- 3 H]-inositol-prelabelled hVSMC. Our findings invoke a role for ET which extends beyond the paracrine regulation by peptide synthesized and secreted by endothelial cells. We propose that VSMC-synthesized ET may function in an autocrine manner to regulate both tone and structural modelling of vasculature.

Stretch affects phenotype and proliferation of vascular smooth muscle cells

The exertion of periodic dynamic strain on the arterial wall is hypothesized to be relevant to smooth muscle cell morphology and function. This study has investigated the effect of cyclic mechanical stretching on rabbit aortic smooth muscle cell proliferation and expression of contractile phenotype protein markers. Cells were cultured on flexible-bottomed dishes and cyclic stretch was applied (frequency 30 cycles/min, 15% elongation) using a Flexercell Strain unit. Cyclic stretch potentiated smooth muscle cell proliferation in serum-activated cultures but not in cultures maintained in 0.5% fetal calf serum. Stretching induced a serum-independent increase of h-caldesmon expression and this effect was reversible following termination of mechanical stimulation. Strain was without effect on smooth muscle myosin or calponin expression. In cells grown on laminin stretch-induced h-caldesmon expression was more prominent than in cells cultured on collagen types I and IV, poly-L-lysine and gelatin. These data suggest that cyclic mechanical stimulation possesses dual effect on vascular smooth muscle cell phenotype characteristics since it: 1) potentiates proliferation, an attribute of a dedifferentiated phenotype; and 2) increases expression of h-caldesmon considered a marker of a differentiated smooth muscle cell state.

Activation of human peripheral monocytes by angiotensin II

This study has investigated the ability of the vasoconstrictor peptide angiotensin II to activate human peripheral blood monocytes. Activation was monitored by measuring both the release of tumor necrosis factor α from monocytes and their adhesion to monolayers of human endothelial cells. Angiotensin II‐elicited activation of monocytes was dose‐dependent (half‐maxiinally effective concentration ≈ 0.2 nM), saturable (maximally effective concentration ≈ 5 nM), and sensitive to inhibition by the angiotensin type 1 receptor antagonist ZD 7155. Such direct actions imply that angiotensin II is an important candidate stimulus for the subendothelial infiltration of monocytes observed in atherogenesis and hypertension.

PDGF-BB increases endothelial migration and cord movements during angiogenesis in vitro

To explore direct effects of platelet‐derived growth factor (PDGF) on endothelial cells during angiogenesis in vitro, we have used cloned bovine aortic endothelial cells that spontaneously form cord structures. Recently we have shown that cells forming these endothelial cords express PDGF β‐receptors and that PDGF‐BB can contribute to cellular proliferation and cord formation. In this study we investigated whether PDGF‐induced cellular migration might also contribute to endothelial repair and angiogenesis in vitro.

Stimulation of autocrine platelet- derived growth factor AA-homodimer and transforming growth factor β in vascular smooth muscle cells

We have investigated the interrelationship between growth factors and vasoconstrictor peptides in terms of their possible paracrine/autocrine regulation of vascular smooth muscle cell differentiation/proliferation. Responses of quiescent cells from spontaneously hypertensive and Wistar-Kyoto rats to stimulation with a selected number of growth factors- and vasoconstrictor peptides were established (induction of mRNA as well as secretion of immunoreactive peptides). A single exposure of quiescent vascular smooth muscle cells to the vasoconstrictor peptides Angiotensin II and Endothelin-1 (10(-8) M each) resulted in a prolonged induction of platelet- derived growth factor A-chain and transforming growth factor beta transcripts (maximal at 5-6 hrs poststimulatory). The interrelationship between platelet- derived growth factor AA and transforming growth factor beta was investigated in experiments using the pure peptides individually for stimulation of mRNA and peptide secretion. Both growth factors enhanced their own and one anothers transcript expression. The results demonstrated that in spontaneously hypertensive rats, an established animal model of hypertension, the steady state balance of this set of growth factors may be disturbed. Defects involved may be attributable to alterations in the secretory machinery and/or amount of autocrine growth factor produced.

Modulation of extracellular matrix by angiotensin II : stimulated glycoconjugate synthesis and growth in vascular smooth muscle cells

A role for angiotensin II (Ang II) in the pathogenesis of hypertension and atherosclerosis was studied using cultured vascular smooth muscle cells from spontaneously hypertensive rats. Chronic exposure of vascular smooth muscle cells, cultured in the presence of 1% plasma-derived serum, to Ang II resulted in a dose-dependent stimulation in growth and incorporation of radiolabeled matrix precursors into extracellular matrix-associated glycoconjugate material. The hormone also stimulated the incorporation of [3H]glycine into extracellular matrix glycoproteins and proteoglycans synthesized by cultures rendered quiescent by maintenance on serum-free medium for 48 h prior to exposure to Ang II. This was negated in the presence of saralasin. In quiescent cultures, a single exposure to angiotensin induced a rapid induction of mRNA coding for the extracellular matrix glycoprotein thrombospondin. Similar results were obtained with cells maintained on medium containing 1% plasma-derived serum; however, the levels of induction were reduced by this procedure. This study demonstrated that Ang II was capable of stimulating both growth and matrix elaboration by cultured vascular smooth muscle cells. These observations are indicative of a pathophysiological role for the vasoconstrictor peptide, which may contribute significantly to the development of hypertension.

Induction of endothelin secretion by angiotensin II: Effects on growth and synthetic activity of vascular smooth muscle cells

Angiotensin II induces the synthesis and secretion of endothelin by cultured rat vascular smooth muscle cells. Previous studies demonstrate that angiotensin II also activates the synthesis of extracellular matrix-associated glycoconjugates (glycopeptides and proteoglycans) by cultured smooth muscle cells. Furthermore, in culture medium containing mitogen-depleted, plasma-derived serum (1.0%), angiotensin II stimulates the growth of rat smooth muscle cells. Therefore, the influence of endothelin-1 (ET-1) on the growth and matrix elaboration by cultured rat smooth muscle cells was studied to assess its contribution to the stimulatory activity of angiotensin II. In quiescent cultures of smooth muscle cells, no stimulation of the incorporation of [3H]thymidine into DNA by ET-1 was apparent, even at maximal doses (10(-7) M). Under the same conditions, the synthesis of extracellular matrix glycoconjugate material was not enhanced by ET-1, unlike angiotensin II. In cultures maintained in medium containing 1.0% plasma-derived serum, ET-1 was incapable of stimulating either proliferation or incorporation of [3H]thymidine into DNA whereas angiotensin II stimulated both activities. ET-1, however, did induce the expression of growth factor and thrombospondin genes in quiescent smooth muscle cultures. The data suggest that growth stimulation of smooth muscle cells by angiotensin II does not occur as a consequence of stimulated ET-1 production.

Functional Aspects of Vascular Tenascin-C Expression

The arterial tenascin C expression in vivo and in vitro has been studied using immunohistochemistry. The functional relevance of localized tenascin C expression was assessed in vitro using various human cell types involved in the progression of vascular disease. Normotensive and hypertensive rats exhibited age-dependent patterns of vascular (aorta) tenascin expression, but the lumen-to-media-directed progression of tenascin induction was accelerated in hypertensive rats. Tenascin-rich neointimal lesions (spontaneous) were observed at branching sites of aorta from aged (80 weeks) hypertensive rats. Subendothelial tenascin foci contained lipid-laden smooth muscle cells and monocytes/macrophages. Medial tenascin foci encaged smooth muscle cells which synthesized DNA. Tenascin was expressed both in vivo and in vitro by endothelial and smooth muscle cells but not by monocytes/macrophages; angiotensin II, oxidized-low density lipoprotein and transforming growth factor beta 1 induced expression of tenascin transcripts and glycoprotein in vitro. Endothelial and smooth muscle cells, but not monocytes, adhered to tenascin substrata. Tenascin reduced focal adhesion integrity in confluent endothelial and smooth muscle cell cultures. Angiotensin II-induced migration of endothelial and smooth muscle cells was accompanied by tenascin deposition within extracellular matrix migration trails. Tenascin may function both as a defense against monocyte invasion and medial smooth muscle replication, as well as a substratum for directed endothelial and smooth muscle cell migration.

Peptide vasoconstrictors, vessel structure, and vascular smooth-muscle proliferation

The peptide vasoconstrictors angiotensin II (Ang II) and endothelin-1 (ET-1), originally thought to derive exclusively from the plasma renin-angiotensin system and vascular endothelium, respectively, have been demonstrated to be produced independently of such sources. Local tissue angiotensin-generating systems are well documented, and endothelin production has been demonstrated for a variety of nonendothelial cells, including vascular smooth-muscle cells (VSMC). There is increasing evidence from in vitro studies that local production of these vasoconstrictor peptides may contribute to blood vessel homeostasis and the development of vascular pathologies. Results obtained from pharmaceutical intervention in humans and animals of these systems strongly support this hypothesis. In addition to their vasoconstrictor properties, Ang II and ET-1 act as potent biological effectors. In vitro, both vasoconstrictor peptides appear to modulate the activity of autocrine feedback loops in VSMC. The activity of these feedback loops in vivo may represent a central mechanism for regulation and phenotypic differentiation of this cell type. The best-recognized autocrine feedback loops of VSMC are constituted by platelet-derived growth factor and transforming growth factor-beta, both of which are influenced by the action of Ang II and ET-1. Because both vasoconstrictors (via their induction of autocrine growth modulators) may influence the composition of the extracellular matrix of VSMC, the effects of the peptide vasoconstrictors on the (auto-) regulated feedback loops are of long-term structural importance. Ang II and ET-1 promote the synthesis and secretion of the glycoproteins thrombospondin, fibronectin, and tenascin.(ABSTRACT TRUNCATED AT 250 WORDS)