Abstract 370: Matrix Metalloproteinase-3 Regulates Arterial Calcification

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Tonghui Lin ◽  
Xue-lin Wang ◽  
Yujun Cai ◽  
Sara L Zettervall ◽  
Raul J Guzman
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Knockout Mice ◽  
Muscle Cells ◽  
Calcium Deposition ◽  
Arterial Calcification ◽  
Wild Type ◽  
Medial Artery Calcification ◽  
Medial Artery

Objective: Medial artery calcification is associated with increased cardiovascular morbidity and mortality in patients with peripheral artery disease (PAD). It is highly prevalent in diabetes and chronic kidney disease, and it is an important risk factor for cardiovascular events. Accumulating evidences suggest that matrix degrading enzymes are prominently involved in this process. Clinical studies have shown that the matrix metallopeptidase3 (MMP-3, stromelysin-1) is correlated with prevalent arterial calcification in high risk human populations. In this study, we sought to determine whether reducing MMP-3 activity could decrease arterial calcification in smooth muscle cells, organ culture, and in vivo models. Methods and Results: Confluent human aortic smooth muscle cells (HASMCs) were cultured in calcification medium containing high phosphate (Pi) for 7 days in the absence or presence of an MMP-3 specific inhibitor. We found that adding MMP-3 inhibitor dose-dependently reduced Pi-induced calcium deposition as demonstrated by the O-cresolphthalein complexone assay. To further determine the importance of MMP-3 in arterial calcification, we utilized ex vivo aortic rings. In aortic rings from MMP-3 knockout mice, Pi-induced medial calcification was significantly reduced compared with wild-type mice. We next evaluated the effects of MMP-3 deletion in vivo . 12-week-old MMP-3 knockout and wild type mice were injected with 8mg/kg cholecalciferol and arteries were collected after 8 days, Calcium levels were significantly lower in MMP-3 knockout mice. Deletion of MMP-3 also reduced serum calcium levels, but not changed serum phosphate level. In concurrent studies, we showed by immunohistochemical staining that MMP-3 was highly expressed in calcified lesion from human tibial arteries. Conclusion: Together these findings suggest that MMP-3 promotes medial artery calcification, and that it may serve as a potential therapeutic target aimed at improving the poor outcomes of our patients with PAD.

scholarly journals Nano-Hydroxyapatite Accelerates Vascular Calcification by Inhibiting Lysosomal Acidification in Smooth Muscle Cells.

2021 ◽  
Author(s):  
Qi Liu ◽  
Yi Luo ◽  
Yun Zhao ◽  
Pingping Xiang ◽  
Jinyun Zhu ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Calcification ◽  
Chemical Properties ◽  
Muscle Cells ◽  
Calcium Deposition ◽  
Rapid Progression ◽  
Autophagic Flux

Abstract Background: Vascular calcification (VC) is a common characteristic of aging, diabetes, chronic renal failure, and atherosclerosis. The basic component of VC is hydroxyapatite (HAp). Nano-sized HAp (nHAp) has been identified as the initiator of pathological calcification of vasculature. However, whether nHAp can induce calcification in vivo and the mechanism of nHAp in the progression of VC remains unclear.Results: We discovered that nHAp existed both in vascular smooth muscle cells (VSMCs) and their extracellular matrix (ECM) in the calcified arteries from patients. Synthetic nHAp had similar morphological and chemical properties as natural nHAp recovered from calcified artery. nHAp induced rapid progression of VC by stimulating osteogenic differentiation and accelerating mineralization of VSMCs in vitro. Synthetic nHAp could also directly induce VC in vivo. Mechanistically, nHAp was internalized into lysosome, which impaired lysosome vacuolar H+-ATPase for its acidification, therefore blocked autophagic flux in VSMCs. The accumulated autophagosomes and autolysosomes were converted into calcium-containing exosomes which were secreted into ECM and accelerated vascular calcium deposit. Inhibition of exosome release in VSMCs decreased calcium deposition. Conclusions: Our results illustrated a novel mechanism of nHAp-induced vascular calcification. Understanding the role of nHAp in autophagy-lysosome-exosome pathway in SMCs could have great clinical significance in preventing the progression of VC.

scholarly journals Cell-Specific Gene Deletion Reveals the Antithrombotic Function of COX1 and Explains the Vascular COX1/Prostacyclin Paradox

2019 ◽  
Vol 125 (9) ◽  
pp. 847-854 ◽  
Author(s):  
Jane A. Mitchell ◽  
Fisnik Shala ◽  
Youssef Elghazouli ◽  
Timothy D. Warner ◽  
Carles Gaston-Massuet ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Knockout Mice ◽  
Muscle Cells ◽  
Protective Role ◽  
Specific Gene ◽  
Prostacyclin Receptor ◽  
First Time ◽  
Prostacyclin Production

Rationale: Endothelial cells (ECs) and platelets, which respectively produce antithrombotic prostacyclin and prothrombotic thromboxane A 2 , both express COX1 (cyclooxygenase1). Consequently, there has been no way to delineate any antithrombotic role for COX1-derived prostacyclin from the prothrombotic effects of platelet COX1. By contrast, an antithrombotic role for COX2, which is absent in platelets, is straightforward to demonstrate. This has resulted in an incomplete understanding of the relative importance of COX1 versus COX2 in prostacyclin production and antithrombotic protection in vivo. Objective: We sought to identify the role, if any, of COX1-derived prostacyclin in antithrombotic protection in vivo and compare this to the established protective role of COX2. Methods and Results: We developed vascular-specific COX1 knockout mice and studied them alongside endothelial-specific COX2 knockout mice. COX1 immunoreactivity and prostacyclin production were primarily associated with the endothelial layer of aortae; freshly isolated aortic ECs released >10-fold more prostacyclin than smooth muscle cells. Moreover, aortic prostacyclin production, the ability of aortic rings to inhibit platelet aggregation and plasma prostacyclin levels were reduced when COX1 was knocked out in ECs but not in smooth muscle cells. When thrombosis was measured in vivo after FeCl 3 carotid artery injury, endothelial COX1 deletion accelerated thrombosis to a similar extent as prostacyclin receptor blockade. However, this effect was lost when COX1 was deleted from both ECs and platelets. Deletion of COX2 from ECs also resulted in a prothrombotic phenotype that was independent of local vascular prostacyclin production. Conclusions: These data demonstrate for the first time that, in healthy animals, endothelial COX1 provides an essential antithrombotic tone, which is masked when COX1 activity is lost in both ECs and platelets. These results help us define a new 2-component paradigm wherein thrombotic tone is regulated by both COX1 and COX2 through complementary but mechanistically distinct pathways.

scholarly journals Calcium Responses to Thyrotropin-Releasing Hormone, Gonadotropin-Releasing Hormone and Somatostatin in Phospholipase Cβ3 Knockout Mice

2001 ◽  
Vol 15 (1) ◽  
pp. 125-135
Author(s):  
Valerie A. Romoser ◽  
Thomas K. Graves ◽  
Dianqing Wu ◽  
Huiping Jiang ◽  
Patricia M. Hinkle
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Intracellular Calcium ◽  
Knockout Mice ◽  
Calcium Transient ◽  
Muscle Cells ◽  
Pituitary Cells ◽  
Wild Type ◽  
Releasing Hormone ◽  
In Cells

Abstract These studies examined the importance of phospholipase Cβ (PLCβ) in the calcium responses of pituitary cells using PLCβ3 knockout mice. Pituitary tissue from wild-type mice contained PLCβ1 and PLCβ3 but not PLCβ2 or PLCβ4. Both Gαq/11 and Gβγ can activate PLCβ3, whereas only Gαq/11 activates PLCβ1 effectively. In knockout mice, PLCβ3 was absent, PLCβ1 was not up-regulated, and PLCβ2 and PLCβ4 were not expressed. Since somatostatin inhibited influx of extracellular calcium in pituitary cells from wild-type and PLCβ3 knockout mice, the somatostatin signal pathway was intact. However, somatostatin failed to increase intracellular calcium in pituitary cells from either wild-type or knockout mice under a variety of conditions, indicating that it did not stimulate PLCβ3. In contrast, somatostatin increased intracellular calcium in aortic smooth muscle cells from wild-type mice, although it evoked no calcium response in cells from PLCβ3 knockout animals. These results show that somatostatin, like other Gi/Go-linked hormones, can stimulate a calcium transient by activating PLCβ3 through Gβγ, but this response does not normally occur in pituitary cells. The densities of Gi and Go, as well as the relative concentrations of PLCβ1 and PLCβ3, were similar in cells that responded to somatostatin with an increase in calcium and pituitary cells. Calcium responses to 1 nm and 1μ m TRH and GnRH were identical in pituitary cells from wild-type and PLCβ3 knockout mice, as were responses to other Gq-linked agonists. These results show that in pituitary cells, PLCβ1 is sufficient to transmit signals from Gq-coupled hormones, whereas PLCβ3 is required for the calcium-mobilizing actions of somatostatin observed in smooth muscle cells.

scholarly journals Receptor-independent Role of Urokinase-Type Plasminogen Activator in Pericellular Plasmin and Matrix Metalloproteinase Proteolysis during Vascular Wound Healing in Mice

1998 ◽  
Vol 140 (1) ◽  
pp. 233-245 ◽  
Author(s):  
Peter Carmeliet ◽  
Lieve Moons ◽  
Mieke Dewerchin ◽  
Steven Rosenberg ◽  
Jean-Marc Herbert ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Neointima Formation ◽  
Wild Type ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

It has been proposed that the urokinase receptor (u-PAR) is essential for the various biological roles of urokinase-type plasminogen activator (u-PA) in vivo, and that smooth muscle cells require u-PA for migration during arterial neointima formation. The present study was undertaken to evaluate the role of u-PAR during this process in mice with targeted disruption of the u-PAR gene (u-PAR−/−). Surprisingly, u-PAR deficiency did not affect arterial neointima formation, neointimal cell accumulation, or migration of smooth muscle cells. Indeed, topographic analysis of arterial wound healing after electric injury revealed that u-PAR−/− smooth muscle cells, originating from the uninjured borders, migrated over a similar distance and at a similar rate into the necrotic center of the wound as wild-type (u-PAR+/+) smooth muscle cells. In addition, u-PAR deficiency did not impair migration of wounded cultured smooth muscle cells in vitro. There were no genotypic differences in reendothelialization of the vascular wound. The minimal role of u-PAR in smooth muscle cell migration was not because of absent expression, since wild-type smooth muscle cells expressed u-PAR mRNA and functional receptor in vitro and in vivo. Pericellular plasmin proteolysis, evaluated by degradation of 125I-labeled fibrin and activation of zymogen matrix metalloproteinases, was similar for u-PAR−/− and u-PAR+/+ cells. Immunoelectron microscopy of injured arteries in vivo revealed that u-PA was bound on the cell surface of u-PAR+/+ cells, whereas it was present in the pericellular space around u-PAR−/− cells. Taken together, these results suggest that binding of u-PA to u-PAR is not required to provide sufficient pericellular u-PA–mediated plasmin proteolysis to allow cellular migration into a vascular wound.

Cannabinoid-1 (CB1) receptors regulate colonic propulsion by acting at motor neurons within the ascending motor pathways in mouse colon

2009 ◽  
Vol 296 (1) ◽  
pp. G119-G128 ◽  
Author(s):  
Andrei Sibaev ◽  
Birol Yüce ◽  
Markus Kemmer ◽  
Luc Van Nassauw ◽  
Ulli Broedl ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Motor Neurons ◽  
Muscle Cells ◽  
Type I ◽  
Wild Type ◽  
Myenteric Neurons ◽  
Circular Smooth Muscle ◽  
Mouse Colon

Cannabinoid-1 (CB1) receptors on myenteric neurons are involved in the regulation of intestinal motility. Our aim was to investigate CB1receptor involvement in ascending neurotransmission in mouse colon and to characterize the involved structures by functional and morphological means. Presence of the CB1receptor was investigated by RT-PCR, and immunohistochemistry was used for colabeling studies. Myenteric reflex responses were initiated by electrical stimulation (ES) at different distances, and junction potentials (JP) were recorded from circular smooth muscle cells by intracellular recording in an unpartitioned and a partitioned recording chamber. In vivo colonic propulsion was tested in wild-type and CB1−/−mice. Immunostaining with the cytoskeletal marker peripherin showed CB1immunoreactivity both on Dogiel type I and type II neurons. Further neurochemical characterization revealed CB1on choline acetyltransferase-, calretinin-, and 5-HT-immunopositive myenteric neurons, but nitrergic neurons appeared immunonegative for CB1immunostaining. Solitary spindle-shaped CB1-immunoreactive cells in between smooth muscle cells lacked specific markers for interstitial cells of Cajal or glial cells. ES elicited neuronally mediated excitatory JP (EJP) and inhibitory JP. Gradual increases in distance resulted in a wave-like EJP with EJP amplitudes being maximal at the location of stimulating electrode 6 and a maximal EJP projection distance of ∼18 mm. The CB1receptor agonist WIN 55,212-2 reduced the amplitude of EJP and was responsible for shortening the oral spreading of the excitatory impulse. In a partitioned chamber, WIN 55,212-2 reduced EJP at the separated oral sites, proving that CB1activation inhibits interneuron-mediated neurotransmission. These effects were absent in the presence of the CB1antagonist SR141716A, which, when given alone, had no effect. WIN 55,212-2 inhibited colonic propulsion in wild-type mice but not in SR141716A-pretreated wild-type or CB1−/−mice. Activation of the CB1receptor modulates excitatory cholinergic neurotransmission in mouse colon by reducing amplitude and spatial spreading of the ascending electrophysiological impulses. This effect on electrophysiological spreading involves CB1-mediated effects on motor neurons and ascending interneurons and is likely to underlie the here reported in vivo reduction in colonic propulsion.

scholarly journals Stromal cells from human long-term marrow cultures are mesenchymal cells that differentiate following a vascular smooth muscle differentiation pathway

Blood ◽  
1993 ◽  
Vol 82 (1) ◽  
pp. 66-76 ◽  
Author(s):  
MC Galmiche ◽  
VE Koteliansky ◽  
J Briere ◽  
P Herve ◽  
P Charbord
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Stromal Cells ◽  
Muscle Cells ◽  
Mesenchymal Cells ◽  
Myoid Cells ◽  
Marrow Cultures

In human long-term marrow cultures connective tissue-forming stromal cells are an essential cellular component of the adherent layer where granulomonocytic progenitors are generated from week 2 onward. We have previously found that most stromal cells in confluent cultures were stained by monoclonal antibodies directed against smooth muscle- specific actin isoforms. The present study was carried out to evaluate the time course of alpha-SM-positive stromal cells and to search for other cytoskeletal proteins specific for smooth muscle cells. It was found that the expression of alpha-SM in stromal cells was time dependent. Most of the adherent spindle-shaped, vimentin-positive stromal cells observed during the first 2 weeks of culture were alpha- SM negative. On the contrary, from week 3 to week 7, most interdigitated stromal cells contained stress fibers whose backbone was made of alpha-SM-positive microfilaments. In addition, in confluent cultures, other proteins specific for smooth muscle were detected: metavinculin, h-caldesmon, smooth muscle myosin heavy chains, and calponin. This study confirms the similarity between stromal cells and smooth muscle cells. Moreover, our results reveal that cells in vivo with the phenotype closest to that of stromal cells are immature fetal smooth muscle cells and subendothelial intimal smooth muscle cells; a cell subset with limited development following birth but extensively recruited in atherosclerotic lesions. Stromal cells very probably derive from mesenchymal cells that differentiate along this distinctive vascular smooth muscle cell pathway. In humans, this differentiation seems crucial for the maintenance of granulomonopoiesis. These in vitro studies were completed by examination of trephine bone marrow biopsies from adults without hematologic abnormalities. These studies revealed the presence of alpha-SM-positive cells at diverse locations: vascular smooth muscle cells in the media of arteries and arterioles, pericytes lining capillaries, myoid cells lining sinuses at the abluminal side of endothelial cells or found within the hematopoietic logettes, and endosteal cells lining bone trabeculae. More or less mature cells of the granulocytic series were in intimate contact with the thin cytoplasmic extensions of myoid cells. Myoid cells may be the in vivo counterpart of stromal cells with the above-described vascular smooth muscle phenotype.

scholarly journals Induction of cyclooxygenase-2 in rat vascular smooth muscle cells in vitro and in vivo.

1994 ◽  
Vol 269 (11) ◽  
pp. 8504-8509
Author(s):  
K.A. Pritchard ◽  
M.K. O'Banion ◽  
J.M. Miano ◽  
N. Vlasic ◽  
U.G. Bhatia ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Cyclooxygenase 2

Angiotensin II modulates frizzled-2 receptor expression in rat vascular smooth muscle cells

2005 ◽  
Vol 108 (6) ◽  
pp. 523-530 ◽  
Author(s):  
Giovanna CASTOLDI ◽  
Serena REDAELLI ◽  
Willy M. M. van de GREEF ◽  
Cira R. T. di GIOIA ◽  
Giuseppe BUSCA ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Angiotensin Ii ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Muscle Cells ◽  
Ang Ii ◽  
Aortic Smooth Muscle

Ang II (angiotensin II) has multiple effects on vascular smooth muscle cells through the modulation of different classes of genes. Using the mRNA differential-display method to investigate gene expression in rat aortic smooth muscle cells in culture in response to 3 h of Ang II stimulation, we observed that Ang II down-regulated the expression of a member of the family of transmembrane receptors for Wnt proteins that was identified as Fzd2 [Fzd (frizzled)-2 receptor]. Fzds are a class of highly conserved genes playing a fundamental role in the developmental processes. In vitro, time course experiments demonstrated that Ang II induced a significant increase (P<0.05) in Fzd2 expression after 30 min, whereas it caused a significant decrease (P<0.05) in Fzd2 expression at 3 h. A similar rapid up-regulation after Ang II stimulation for 30 min was evident for TGFβ1 (transforming growth factor β1; P<0.05). To investigate whether Ang II also modulated Fzd2 expression in vivo, exogenous Ang II was administered to Sprague–Dawley rats (200 ng·kg−1 of body weight·min−1; subcutaneously) for 1 and 4 weeks. Control rats received normal saline. After treatment, systolic blood pressure was significantly higher (P<0.01), whereas plasma renin activity was suppressed (P<0.01) in Ang II- compared with the saline-treated rats. Ang II administration for 1 week did not modify Fzd2 expression in aorta of Ang II-treated rats, whereas Ang II administration for 4 weeks increased Fzd2 mRNA expression (P<0.05) in the tunica media of the aorta, resulting in a positive immunostaining for fibronectin at this time point. In conclusion, our data demonstrate that Ang II modulates Fzd2 expression in aortic smooth muscle cells both in vitro and in vivo.

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