Expanding the Reactive Sulfur Metabolome: Intracellular and Efflux Measurements of Small Oxoacids of Sulfur (SOS) and H2S in Human Primary Vascular Cell Culture

Hydrogen sulfide (H2S) is an endogenous signaling molecule which is important for cardiovascular health, but its mechanism of action remains poorly understood. Here, we report measurements of H2S as well as its oxidized metabolites, termed small oxoacids of sulfur (SOS = HSOH and HOSOH), in four human primary vascular cell lines: smooth muscle and endothelial cells derived from both human arterial and coronary tissues. We use a methodology that targets small molecular weight sulfur species; mass spectrometric analysis allows for species quantification to report cellular concentrations based on an H2S calibration curve. The production of H2S and SOS is orders of magnitude higher in smooth muscle (nanomolar) as compared to endothelial cell lines (picomolar). In all the primary lines measured, the distributions of these three species were HOSOH >H2S > HSOH, with much higher SOS than seen previously in non-vascular cell lines. H2S and SOS were effluxed from smooth muscle cells in higher concentrations than endothelial cells. Aortic smooth muscle cells were used to examine changes under hypoxic growth conditions. Hypoxia caused notable increases in HSOH and ROS, which we attribute to enhanced sulfide quinone oxidase activity that results in reverse electron transport.

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Metabolic cooperation between vascular endothelial cells and smooth muscle cells in co-culture: changes in low density lipoprotein metabolism.

The Journal of Cell Biology ◽

10.1083/jcb.101.3.871 ◽

1985 ◽

Vol 101 (3) ◽

pp. 871-879 ◽

Cited By ~ 52

Author(s):

P F Davies ◽

G A Truskey ◽

H B Warren ◽

S E O'Connor ◽

B H Eisenhaure

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Rate Constant ◽

Low Density Lipoprotein ◽

Lipoprotein Metabolism ◽

Density Lipoprotein ◽

Muscle Cells ◽

Low Density ◽

Vascular Cell

A microcarrier co-culture system for aortic endothelial cells and smooth muscle cells (SMCs) was developed as a model for metabolic interactions between cells of the vessel wall. Low density lipoprotein (LDL) metabolism in SMCs was significantly influenced by co-culture with endothelium. The numbers of high affinity receptors for LDL was increased more than twofold (range, 2.1-5.6), with concomitant increases in LDL receptor-mediated endocytosis and degradation. These effects reached a plateau at an endothelial cell/SMC ratio of 1. Kinetic analysis of the endocytic pathway for LDL in SMCs indicated that, in co-culture with endothelium, there was no alteration in the binding affinity of LDL to its receptors but that the internalization rate constant declined and the rate constant for degradation increased. This analysis suggested that the formation and migration of endocytic vesicles was the rate-limiting step of enhanced LDL metabolism under co-culture conditions. Two mechanisms by which endothelial cells influenced smooth muscle LDL metabolism were identified. First, mitogen(s) derived from endothelial cells stimulated entry of SMCs into the growth cycle, and the changes in LDL metabolism occurred as a consequence of G1-S transition. Second, SMC lipoprotein metabolism was stimulated in the absence of mitogens by a low molecular weight (less than 3,500) factor or factors. Co-culture was a required condition for the latter effect, suggesting that the mediator(s) may be unstable or that cell-cell communication was necessary for expression. These results (a) demonstrate that vascular cell interactions can modify LDL metabolism in SMCs, (b) provide some insights into the mechanisms responsible, and (c) identify co-culture as an experimental approach appropriate to certain aspects of vascular cell biology.

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ACE2/Ang-(1-7)/Mas1 axis and the vascular system: vasoprotection to COVID-19-associated vascular disease

Clinical Science ◽

10.1042/cs20200480 ◽

2021 ◽

Vol 135 (2) ◽

pp. 387-407

Author(s):

Jithin Kuriakose ◽

Augusto C. Montezano ◽

Rhian M. Touyz

Keyword(s):

Cardiovascular Disease ◽

Endothelial Cells ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Cardiovascular Health ◽

Vascular System ◽

Prognostic Significance ◽

Muscle Cells ◽

Ang Ii ◽

Enzymatic Function

Abstract The two axes of the renin–angiotensin system include the classical ACE/Ang II/AT1 axis and the counter-regulatory ACE2/Ang-(1-7)/Mas1 axis. ACE2 is a multifunctional monocarboxypeptidase responsible for generating Ang-(1-7) from Ang II. ACE2 is important in the vascular system where it is found in arterial and venous endothelial cells and arterial smooth muscle cells in many vascular beds. Among the best characterized functions of ACE2 is its role in regulating vascular tone. ACE2 through its effector peptide Ang-(1-7) and receptor Mas1 induces vasodilation and attenuates Ang II-induced vasoconstriction. In endothelial cells activation of the ACE2/Ang-(1-7)/Mas1 axis increases production of the vasodilator’s nitric oxide and prostacyclin’s and in vascular smooth muscle cells it inhibits pro-contractile and pro-inflammatory signaling. Endothelial ACE2 is cleaved by proteases, shed into the circulation and measured as soluble ACE2. Plasma ACE2 activity is increased in cardiovascular disease and may have prognostic significance in disease severity. In addition to its enzymatic function, ACE2 is the receptor for severe acute respiratory syndrome (SARS)-coronavirus (CoV) and SARS-Cov-2, which cause SARS and coronavirus disease-19 (COVID-19) respectively. ACE-2 is thus a double-edged sword: it promotes cardiovascular health while also facilitating the devastations caused by coronaviruses. COVID-19 is associated with cardiovascular disease as a risk factor and as a complication. Mechanisms linking COVID-19 and cardiovascular disease are unclear, but vascular ACE2 may be important. This review focuses on the vascular biology and (patho)physiology of ACE2 in cardiovascular health and disease and briefly discusses the role of vascular ACE2 as a potential mediator of vascular injury in COVID-19.

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Plasminogen Activator Inhibitor-1 Expression in Human Liver and Healthy or Atherosclerotic Vessel Walls

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648809 ◽

1994 ◽

Vol 72 (01) ◽

pp. 044-053 ◽

Cited By ~ 76

Author(s):

N Chomiki ◽

M Henry ◽

M C Alessi ◽

F Anfosso ◽

I Juhan-Vague

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Plasminogen Activator ◽

Human Liver ◽

Plasminogen Activator Inhibitor ◽

Muscle Cells ◽

Vessel Walls ◽

Activator Inhibitor ◽

Pai 1

SummaryIndividuals with elevated levels of plasminogen activator inhibitor type 1 are at risk of developing atherosclerosis. The mechanisms leading to increased plasma PAI-1 concentrations are not well understood. The link observed between increased PAI-1 levels and insulin resistance has lead workers to investigate the effects of insulin or triglyceride rich lipoproteins on PAI-1 production by cultured hepatocytes or endothelial cells. However, little is known about the contribution of these cells to PAI-1 production in vivo. We have studied the expression of PAI-1 in human liver sections as well as in vessel walls from different territories, by immunocytochemistry and in situ hybridization.We have observed that normal liver endothelial cells expressed PAI-1 while parenchymal cells did not. However, this fact does not refute the role of parenchymal liver cells in pathological states.In healthy vessels, PAI-1 mRNA and protein were detected primarily at the endothelium from the lumen as well as from the vasa vasorum. In normal arteries, smooth muscle cells were able to produce PAI-1 depending on the territory tested. In deeply altered vessels, PAI-1 expression was observed in neovessels scattering the lesions, in some intimal cells and in smooth muscle cells. Local increase PAI-1 mRNA described in atherosclerotic lesions could be due to the abundant neovascularization present in the lesion as well as a raised expression in smooth muscle cells. The increased PAI-1 in atherosclerosis could lead to fibrin deposit during plaque rupture contributing further to the development and progression of the lesion.

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Thrombin Inhibition and Thrombin Generation by Cultured Aortic Endothelial and Smooth Muscle Cells from Minipig

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657227 ◽

1982 ◽

Vol 48 (01) ◽

pp. 101-103 ◽

Cited By ~ 1

Author(s):

B Kirchhof ◽

J Grünwald

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Smooth Muscle Cells ◽

Vessel Wall ◽

Thrombin Generation ◽

Muscle Cells ◽

Thrombin Inhibition ◽

Generating Capacity ◽

Wall Interaction ◽

Cells Cultured

SummaryEndothelial and smooth muscle cells cultured from minipig aorta were examined for their inhibitory activity on thrombin and for their thrombin generating capacity.Endothelial cells showed both a thrombin inhibition and an activation of prothrombin in the presence of Ca++, which was enhanced in the presence of phospholipids. Smooth muscle cells showed an activation of prothrombin but at a lower rate. Both coagulation and amidolytic micro-assays were suitable for studying the thrombin-vessel wall interaction.

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Vascular Smooth Muscle Cells Inhibit the Plasminogen Activators Secreted by Endothelial Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661265 ◽

1985 ◽

Vol 53 (02) ◽

pp. 165-169 ◽

Cited By ~ 24

Author(s):

Walter E Laug

Keyword(s):

Endothelial Cells ◽

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Conditioned Medium ◽

Vascular Smooth Muscle Cells ◽

Inhibitory Activity ◽

Muscle Cells ◽

Plasminogen Activators ◽

Bovine Endothelial Cells

SummaryTPure cultures of bovine endothelial cells (EC) produce and secrete large amounts of plasminogen activators (PA). Cocultivation of EC with vascular smooth muscle cells (SMC) resulted in a significant decrease of PA activities secreted by the EC, whereas the cellular PA activities remained unaffected. Secreted PA activities were absent in the growth medium as long as the SMC to EC ratio was 2:1 or higher. The PA inhibitory activity of the SMC was rapid and cell-to-cell contact was not necessary.The PA inhibitory activity was present in homogenates of SMC as well as in the medium conditioned by them but not in the extracellular matrix elaborated by these cells. Serum free medium conditioned by SMC neutralized both tissue type (t-PA) and urokinase like (u-PA) plasminogen activators. Gel electrophoretic analysis of SMC conditioned medium followed by reverse fibrin autography demonstrated PA inhibitory activities in the molecular weight (Mr) range of 50,000 to 52,000 similar to those present in media conditioned by bovine endothelial cells or fibroblasts. Regular fibrin zymography of SMC conditioned medium incubated with u-PA or t-PA revealed the presence of a component with a calculated approximate Mr of 45,000 to 50,000 which formed SDS resistant complexes with both types of PA.These data demonstrate that vascular SMC produce and secrete (a) inhibitor(s) of PAs which may influence the fibrinolytic potential of EC.

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