Collagen-induced Changes Research Articles

Increased protein phosphatase 5 expression in inflammation-induced left ventricular dysfunction in rats

Titin phosphorylation contributes to left ventricular (LV) diastolic dysfunction. The independent effects of inflammation on the molecular pathways that regulate titin phosphorylation are unclear. We investigated the effects of collagen-induced inflammation and subsequent tumor necrosis factor-α (TNF-α) inhibition on mRNA expression of genes involved in regulating titin phosphorylation in 70 Sprague-Dawley rats. LV diastolic function was assessed with echocardiography. Circulating inflammatory markers were quantified by enzyme-linked immunosorbent assay and relative LV gene expression was assessed by Taqman® polymerase chain reaction. Differences in normally distributed variables between the groups were determined by two-way analysis of variance (ANOVA), followed by Tukey post-hoc tests. For non-normally distributed variables, group differences were determined by Kruskal-Wallis tests. Collagen inoculation increased LV relative mRNA expression of vascular cell adhesion molecule 1 (VCAM1), pentraxin 3 (PTX3), and inducible nitric oxide synthase (iNOS) compared to controls, indicating local microvascular inflammation. Collagen inoculation decreased soluble guanylate cyclase alpha-2 (sGCα2) and soluble guanylate cyclase beta-2 (sGCβ2) expression, suggesting downregulation of nitric oxide-soluble guanylate cyclase-cyclic guanosine monophosphate (NO-sGC-cGMP) signaling. Inhibiting TNF-α prevented collagen-induced changes in VCAM1, iNOS, sGCα2 and sGCβ2 expression. Collagen inoculation increased protein phosphatase 5 (PP5) expression. Like LV diastolic dysfunction, increased PP5 expression was not prevented by TNF-α inhibition. Inflammation-induced LV diastolic dysfunction may be mediated by a TNF-α-independent increase in PP5 expression and dephosphorylation of the N2-Bus stretch element of titin, rather than by TNF-α-induced downregulation of NO-sGC-cGMP pathway-dependent titin phosphorylation. The steady rise in number of patients with inflammation-induced diastolic dysfunction, coupled with low success rates of current therapies warrants a better understanding of the systemic signals and molecular pathways responsible for decreased titin phosphorylation in development of LV diastolic dysfunction. The therapeutic potential of inhibiting PP5 upregulation in LV diastolic dysfunction requires investigation.

Cholesterol crystallization in human atherosclerosis is triggered in smooth muscle cells during the transition from fatty streak to fibroatheroma.

Recent studies have shown that in addition to being major constituents of the atheromatous core, solid cholesterol crystals (CCs) promote atherosclerotic lesion development and rupture by causing mechanical damage and exerting cytotoxic and pro-inflammatory effects. These findings suggest that targeting CCs might represent a therapeutic strategy for plaque stabilization. However, little is known about how cholesterol crystallization is initiated in human atherothrombotic disease. Here, we investigated these mechanisms. We performed a thorough immunohistological analysis of non-embedded, minimally processed human aortic tissues, combining polarized light and fluorescence microscopy. We found that CC formation was initiated during the fatty streak to fibroatheroma transition in tight association with the death of intralesional smooth muscle cells (SMCs). Cholesterol-loaded human SMCs were capable of producing CCs in vitro, a process that was enhanced by type I collagen and by inhibition of autophagy and cholesterol esterification. The fibrous transition, which was characterized by increased type I collagen expression, was associated with changes in the expression of autophagy and cholesterol flux-related genes, including a decrease in the autophagic adapter p62 and an increase in the cholesterol intracellular transporter Niemann-Pick C1. Collagen was identified as a potent inducer of these changes in SMCs. Collagen-induced changes in cholesterol metabolism and autophagy flux in smooth muscle foam cells at the fibrolipid transition likely contribute to initiate cholesterol crystallization in human atherosclerosis. Also, our data are in support of a protective role of autophagy against CC formation. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Collagen can selectively trigger a platelet secretory phenotype via glycoprotein VI.

Platelets are not only central actors of hemostasis and thrombosis but also of other processes including inflammation, angiogenesis, and tissue regeneration. Accumulating evidence indicates that these “non classical” functions of platelets do not necessarily rely on their well-known ability to form thrombi upon activation. This suggests the existence of non-thrombotic alternative states of platelets activation. We investigated this possibility through dose-response analysis of thrombin- and collagen-induced changes in platelet phenotype, with regards to morphological and functional markers of platelet activation including shape change, aggregation, P-selectin and phosphatidylserine surface expression, integrin activation, and release of soluble factors. We show that collagen at low dose (0.25 µg/mL) selectively triggers a platelet secretory phenotype characterized by the release of dense- and alpha granule-derived soluble factors without causing any of the other major platelet changes that usually accompany thrombus formation. Using a blocking antibody to glycoprotein VI (GPVI), we further show that this response is mediated by GPVI. Taken together, our results show that platelet activation goes beyond the mechanisms leading to platelet aggregation and also includes alternative platelet phenotypes that might contribute to their thrombus-independent functions.

Extracellular Matrix of Collagen Modulates Intracellular Calcium Handling and Electrophysiological Characteristics of HL-1 Cardiomyocytes With Activation of Angiotensin II Type 1 Receptor

Background Myocardial fibrosis plays a critical role in heart failure, resulting in cardiac structural and electrical remodeling which can induce atrial arrhythmias. Collagen is the major element of fibrosis. However, it is not clear whether collagen can directly regulate the calcium homeostasis and the electrophysiologic characteristics of cardiomyocytes. The aim of this study was to determine the effects of collagen on calcium homeostasis and the electrical properties of atrial cardiomyocytes. Methods and Results HL-1 cardiomyocytes were cultured with and without collagen type I (1 or 10 μg/mL) or losartan (10 μmol/L). Whole-cell clamp, indo-1 fluorescence, and Western blotting were used to evaluate the action potential (AP) and ionic currents, intracellular calcium homeostasis, and calcium regulatory proteins. Compared with the control samples, there was no significant difference in collagen (1 μg/mL)–treated HL-1 cardiomyocytes. However, collagen (10 μg/mL)–treated HL-1 cardiomyocytes exhibited larger intracellular calcium ([Ca 2+] i) transients by 113% and a larger sarcoplasmic reticulum calcium content by 86%. Collagen (10 μg/mL)–treated HL-1 cardiomyocytes had higher expression of sarcoplasmic reticulum ATPase (SERCA2a) and Thr17-phosphorylated phospholamban but similar protein expressions of the Na +/Ca 2+ exchanger and ryanodine receptor. Collagen (10 μg/mL)–treated HL-1 cardiomyocytes (n = 11) had larger AP amplitude (104 ± 5 vs 83 ± 7 mV; P < .05), and shorter 90% of AP duration (25 ± 2 vs 33 ± 2 ms, P < .05) than control cells (n = 11). Moreover, collagen (10 μg/mL)–treated HL-1 cells had larger I to and I Ksus values than control cells. The administration of losartan (10 μmol/L) attenuated collagen-induced changes in [Ca 2+] i transients, [Ca 2+] i stores, AP morphology, ionic currents, SERCA2a, and Thr17-phosphorylated phospholamban expressions. Conclusions This study demonstrates that collagen can directly modulate the calcium dynamics and electrical activities of atrial cardiomyocytes, which are associated with the renin-angiotensin system. These findings suggest a critical role of collagen in electrical remodeling during fibrosis.

Impaired Platelet Activation in Familial High Density Lipoprotein Deficiency (Tangier Disease)

ATP binding cassette transporter A1 (ABCA1) is involved in regulation of intracellular lipid trafficking and export of cholesterol from cells to high density lipoproteins. ABCA1 defects cause Tangier disease, a disorder characterized by absence of high density lipoprotein and thrombocytopenia. In the present study we have demonstrated that ABCA1 is expressed in human platelets and that fibrinogen binding and CD62 surface expression in response to collagen and low concentrations of thrombin, but not to ADP, are defective in platelets from Tangier patients and ABCA1-deficient animals. The expression of platelet membrane receptors such as GPVI, alpha2beta1 integrin, and GPIIb/IIIa, the collagen-induced changes in phosphatidylserine and cholesterol distribution, and the collagen-induced signal transduction examined by phosphorylation of LAT and p72syk and by intracellular Ca2+ mobilization were unaltered in Tangier platelets. The electron microscopy of Tangier platelets revealed reduced numbers of dense bodies and the presence of giant granules typically encountered in platelets from Chediak-Higashi syndrome. Further studies demonstrated impaired release of dense body content in platelets from Tangier patients and ABCA1-deficient animals. In addition, Tangier platelets were characterized by defective surface exposure of dense body and lysosomal markers (CD63, LAMP-1, LAMP-2, CD68) during collagen- and thrombin-induced stimulation and by abnormally high lysosomal pH. We conclude that intact ABCA1 function is necessary for proper maturation of dense bodies in platelets. The impaired release of the content of dense bodies may explain the defective activation of Tangier platelets by collagen and low concentrations of thrombin, but not by ADP.

Collagen-induced changes in the pattern of protein synthesis of fibroblasts.

Cells were cultured on plastic, collagen fibrils or gelatin. General protein synthetic activity of cells did not show any significant difference among the three substrates, whereas the pattern of protein synthesis was substrate-dependent. Profiles of protein synthesis (polypeptide maps) were obtained by subjecting two-dimensional autoradiograms of polyacrylamide gel electrophoresis to a computer-assisted image analyzer. Major polypeptide spots expressed on gelatin were rather like those on plastic. Collagen fibrils caused significant changes in the polypeptides map. Fibroblasts on collagen fibrils produced 364 spots of polypeptides, 26% of which were synthesized specifically on collagen fibrils. The remaining was shared by cells on plastic and was categorized into three groups: (1) polypeptides whose synthesis was up-regulated by collagen fibrils (26% of the total); (2) polypeptides that were expressed equally on both plastic and collagen fibrils (51%); and (3) polypeptides down-regulated by collagen fibrils (23%). A protein with molecular weight of 150 K and an isoelectric point (pI) of 7.3 was one of the collagen-induced and worthy of further analysis. This protein was found to change its pI depending upon the amounts of collagen fibrils and was shown to be located in the mitochondrial fraction.

Eicosanoid-dependent and -independent formation of individual [ 14C]stearoyl-labelled lysophospholipids in collagen-stimulated human platelets

Low level collagen activation of platelets is mediated via the release of arachidonic acid (AA) from membrane phosphoiipids and the formation of thromboxane A 2 (TxA 2). To assess the specific phospholipids undergoing deacylation via phospholipase A 2 thereby providing source(s) of releasable AA, we have measured the individual lysophospholipid formations in platelets prelabelled with [ 14C]stearic acid and incubated with a low level (2 μg/ml) or a high level (10 μg/ml) of collagen in the absence or presence of BW755C, a dual inhibitor of cyclooxygenase and lipoxygenase activities. Collagen activation resulted in the generation of [ 14C]stearoyl-labelled lysophosphatidylinositol (lysoPI), lysophosphatidylcholine (lysoPC), lysophosphatidylethanolamine (lysoPE) and lysophosphatidylserine. BW755C significantly inhibited these collagen-induced changes, suggesting that much of the lysophospholipid, and therefore AA release, was eicosanoid-mediated. At the lower level of collagen, considerable generation of [ 14C]lysoPE was maintained even in the presence of BW755C, suggesting an eicosanoid-independent degradation of phosphatidyl-ethanolamine. The TxA 2-dependent release of AA was also investigated in U-46619-stimulated platelets. This TxA 2 mimetic induced considerable formation of the 14C-labelled lysophospholipids, including lysoPI and lysoPC, but not lysoPE. These results suggest that an eicosanoid-independent degradation of phosphatidylethanolamine via phospholipase A 2 at lower collagen levels may provide a source of the initial AA for conversion to TxA 2 and the subsequent deacylation of phosphatidylinositol, phosphatidylcholine, and also phosphatidylserine.

5‐Hydroxytryptamine and arachidonic acid metabolites modulate extensive platelet activation induced by collagen in cats in vivo

1. The pathways contributing to the platelet adhesion/aggregation reaction elicited by collagen microfibrils, administered to cats in vivo, were analysed. 2. The intra-aortic infusion of collagen (100 micrograms kg-1 in 1 min) caused an extensive activation of platelets, as evidenced by the time-dependent drop of free platelet numbers in whole blood, and the increases of 5-hydroxyindoles (5-HI), 5-hydroxytryptamine (5-HT) and thromboxane B2 (TXB2) levels in plasma, prepared from effluent venous blood sampled from the inferior caval vein. 3. 5-HT2 receptor blockade with ketanserin (0.63 mg kg-1 i.v., 10 min) and cyclo-oxygenase inhibition with aspirin (10 mg kg-1 i.v., 10 min) slightly attenuated the peak reduction of free platelets in whole blood in response to collagen without affecting changes in plasma 5-HI. Aspirin, but not ketanserin, reduced the collagen-induced changes in plasma TXB2, prostaglandin E2 (PGE2) and 6K-PGF1 alpha. 4. Dual TXA2 synthetase inhibition/TXA2-prostaglandin endoperoxide receptor antagonism with ridogrel (5 mg kg-1 i.v., 10 min) halved the drop in free platelets, reduced the release of platelet 5-HI, inhibited the increase in plasma TXB2 and elevated that of 6K-PGF1 alpha and PGE2 in response to collagen. 5. Combined treatment with ketanserin and aspirin reduced the collagen-induced drop of free platelets and the release of platelet 5-HI to a similar extent as ridogrel alone; plasma prostanoids were affected as with aspirin alone. 6. Combined administration of ketanserin and ridogrel virtually eliminated the collagen-induced platelet adhesion/aggregation response and release of 5-HI; prostanoids were affected as with ridogrel alone. 6. Combined administration of ketanserin and ridogrel virtually eliminated the collagen-induced platelet adhesion/aggregation response and release of 5-HI; prostanoids were affected as with ridogrel alone. 7. The results indicate that the interplay between 5-HT and arachidonic acid metabolites is causally involved in the platelet reaction to activation induced by collagen in cats in vivo.

Effect of a Novel Thromboxane A2 Receptor Antagonist, S-145, on Collagen-Induced ECG Changes and Thrombocytopenia in Rodents

The effects of S-145, a newly synthesized thromboxane A2 (TXA2) receptor antagonist, were studied on collagen-induced changes of electrocardiograms (ECG) in rats and thrombocytopenia in rats and mice. Intravenous injection of collagen induced abnormal ECG changes such as elevation or depression of the ST segment, arrhythmia and in severe cases, cardiac arrest. These changes peaked at 3-5 min and lasted for 10 min. S-145 showed remarkable improvement of the ECG changes by both intravenous and oral administration, and the action lasted over 4 hr with 10 mg/kg, p.o. Reference compounds ONO-3708, dazoxiben and aspirin also improved the ECG changes significantly, but ticlopidine was ineffective. S-145 prevented the collagen-induced thrombocytopenia in rats but did not affect the increase in plasma TXB2 levels. S-145 also prevented collagen-induced thrombocytopenia in mice after either intravenous or oral administration in a dose-dependent manner. The efficacy of S-145 was 4-13 times greater than those of the reference compounds, and the duration of action was over 4 hr with 10 mg/kg, p.o. These results indicate that S-145 is a potent, orally active and long-lasting TXA2 receptor antagonist, which will be promising as a drug for thromboembolism and ischemic heart disease caused by platelet activation.

Effect of a novel thromboxane A2 receptor antagonist, S-145, on collagen-induced ECG changes and thrombocytopenia in rodents.

The effects of S-145, a newly synthesized thromboxane, A2 (TXA2) receptor antagonist, were studied on collagen-induced changes of electrocardiograms (ECG) in rats and thrombocytopenia in rats and mice. Intravenous injection of collagen induced abnormal ECG changes such as elevation or depression of the ST segment, arrhythmia and in severe cases, cardiac arrest. These changes peaked at 3-5 min and lasted for 10 min. S-145 showed remarkable improvement of the ECG changes by both intravenous and oral administration, and the action lasted over 4 hr with 10 mg/kg, p.o. Reference compounds ONO-3708, dazoxiben and aspirin also improved the ECG changes significantly, but ticlopidine was ineffective. S-145 prevented the collagen-induced thrombocytopenia in rats but did not affect the increase in plasma TXB2 levels. S-145 also prevented collagen-induced thrombocytopenia in mice after either intravenous or oral administration in a dose-dependent manner. The efficacy of S-145 was 4-13 times greater than those of the reference compounds, and the duration of action was over 4 hr with 10 mg/kg, p.o. These results indicate that S-145 is a potent, orally, active and long-lasting TXA2 receptor antagonist, which will be promising as a drug for thromboembolism and ischemic heart disease caused by platelet activation.

Collagen-induced changes in activity and properties of F X and Xa

Bovine insoluble collagen, devoid of thromboplastic and protease activity, increases the clot-promoting effect of F VII/X complex and purified F X, as well as the esterolytic activity of F Xa. The effects were time dependent and related to the concentration of the factors. Acid-soluble collagen acted similarly on F Xa. Collagenase treatment of the collagen obviates all its effects, and intact helical structure is apparently of critical importance. Collagen-induced electrophoretic changes observed in F X were compared with those produced in the factor when activated by Russell's Viper venom, trypsin, concentrated citrate, and thromboplastin. The results indicated that the site of action of collagen on F X seems to be different from that of RVv or trypsin. Furthermore, collagen augments the activity of the F Xa resulting from activation by RVv or by trypsin. It also changes the electrophoretic profile of F X that has been previously activated by these agents, as well as the thin-layer electrophoresis “fingerprints” of the TCA-soluble products of activation of F X by various systems. These effects of collagen may be highly significant in hemostasis and thrombosis, especially in view of the recognized additional effects on platelets, F XI and F XII.