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Increased fibroblast chymase production mediates procollagen autophagic digestion in volume overload.

ABSTRACT: BACKGROUND:Previous work has identified mast cells as the major source of chymase largely associated with a profibrotic phenotype. We recently reported increased fibroblast autophagic procollagen degradation in a rat model of pure volume overload (VO). Here we demonstrate a connection between increased fibroblast chymase production and autophagic digestion of procollagen in the pure VO of aortocaval fistula (ACF) in the rat. METHODS AND RESULTS:Isolated LV fibroblasts taken from 4 and 12week ACF Sprague-Dawley rats have significant increases in chymase mRNA and chymase activity. Increased intracellular chymase protein is documented by immunocytochemistry in the ACF fibroblasts compared to cells obtained from age-matched sham rats. To implicate VO as a stimulus for chymase production, we show that isolated adult rat LV fibroblasts subjected to 24h of 20% cyclical stretch induces chymase mRNA and protein production. Exogenous chymase treatment of control isolated adult cardiac fibroblasts demonstrates that chymase is internalized through a dynamin-dependent mechanism. Chymase treatment leads to an increased formation of autophagic vacuoles, LC3-II production, autophagic flux, resulting in increased procollagen degradation. Chymase inhibitor treatment reduces cyclical stretch-induced autophagy in isolated cardiac fibroblasts, demonstrating chymase's role in autophagy induction. CONCLUSION:In a pure VO model, chymase produced in adult cardiac fibroblasts leads to autophagic degradation of newly synthesized intracellular procollagen I, suggesting a new role of chymase in extracellular matrix degradation.

SUBMITTER: Fu L

PROVIDER: S-EPMC5198899 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Increased fibroblast chymase production mediates procollagen autophagic digestion in volume overload.

Fu Lianwu L Wei Chih-Chang CC Powell Pamela C PC Bradley Wayne E WE Ahmad Sarfaraz S Ferrario Carlos M CM Collawn James F JF Dell'Italia Louis J LJ

Journal of molecular and cellular cardiology 20160122

<h4>Background</h4>Previous work has identified mast cells as the major source of chymase largely associated with a profibrotic phenotype. We recently reported increased fibroblast autophagic procollagen degradation in a rat model of pure volume overload (VO). Here we demonstrate a connection between increased fibroblast chymase production and autophagic digestion of procollagen in the pure VO of aortocaval fistula (ACF) in the rat.<h4>Methods and results</h4>Isolated LV fibroblasts taken from 4 ...[more]

PMID: 26807691

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Project description:BackgroundThe clinical problem of a "pure volume overload" as in isolated mitral or aortic regurgitation currently has no documented medical therapy that attenuates collagen loss and the resultant left ventricular (LV) dilatation and failure. Here, we identify a potential mechanism related to upregulation of the kallikrein-kinin system in the volume overload of aortocaval fistula (ACF) in the rat.Methodology/principal findingsLV interstitial fluid (ISF) collection, hemodynamics, and echocardiography were performed in age-matched shams and 4 and 15 wk ACF rats. ACF rats had LV dilatation and a 2-fold increase in LV end-diastolic pressure, along with increases in LV ISF bradykinin, myocardial kallikrein and bradykinin type-2 receptor (BK(2)R) mRNA expression. Mast cell numbers were increased and interstitial collagen was decreased at 4 and 15 wk ACF, despite increases in LV ACE and chymase activities. Treatment with the kallikrein inhibitor aprotinin preserved interstitial collagen, prevented the increase in mast cells, and improved LV systolic function at 4 wk ACF. To establish a cause and effect between ISF bradykinin and mast cell-mediated collagen loss, direct LV interstitial bradykinin infusion in vivo for 24 hrs produced a 2-fold increase in mast cell numbers and a 30% decrease in interstitial collagen, which were prevented by BK(2)R antagonist. To further connect myocardial stretch with cellular kallikrein-kinin system upregulation, 24 hrs cyclic stretch of adult cardiomyocytes and fibroblasts produced increased kallikrein, BK(2)R mRNA expressions, bradykinin protein and gelatinase activity, which were all decreased by the kallikrein inhibitor-aprotinin.Conclusions/significanceA pure volume overload is associated with upregulation of the kallikrein-kinin system and ISF bradykinin, which mediates mast cell infiltration, extracellular matrix loss, and LV dysfunction-all of which are improved by kallikrein inhibition. The current investigation provides important new insights into future potential medical therapies for the volume overload of aortic and mitral regurgitation.

Project description:Transforming growth factor-beta 1 (TGF beta 1) initiates a series of signalling events resulting in diverse cellular responses including stimulation of extracellular matrix protein production. In this study we have investigated the role of pertussis toxin-sensitive G-proteins in mediating the effects of TGF beta 1 on fibroblast procollagen metabolism. TGF beta 1 stimulated human fetal lung fibroblast procollagen synthesis and production in a dose-dependent manner which was maximal at 0.5 ng/ml. TGF beta 1 also decreased the proportion of newly synthesized procollagen degraded intracellularly. Pertussis toxin, a G-protein inhibitor, further stimulated TGF beta 1-induced procollagen synthesis and production, but alone it had no effect on fibroblast procollagen metabolism. Addition of indomethacin also potentiated the TGF beta 1-induced increase in procollagen synthesis and production. The effects of pertussis toxin and indomethacin were not additive. Pertussis toxin and indomethacin did not affect the proportion of newly synthesized procollagen degraded intracellularly, either alone or in combination, by control cells. The TGF beta 1-induced decrease in intracellular procollagen degradation was maintained but not further affected by pertussis toxin or indomethacin. TGF beta 1 increased prostaglandin E2 (PGE2) compared with PGE2 production by control cells. Addition of pertussis toxin or indomethacin blocked the TGF beta 1-induced increase in PGE2 production. The TGF beta 1-induced increase in PGE2 preceded the increase in procollagen production. These results demonstrate that TGF beta 1-induced procollagen synthesis by lung fibroblasts is modulated by production of PGE2. Pertussis toxin and indomethacin block the production of PGE2 and enhance the effect of TGF beta 1 on procollagen synthesis. From these data we conclude that the effects of TGF beta 1 on PGE2 production but not procollagen synthesis are mediated via a receptor linked to a pertussis toxin-sensitive G-protein.

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Increased fibroblast chymase production mediates procollagen autophagic digestion in volume overload.

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Increased fibroblast chymase production mediates procollagen autophagic digestion in volume overload.

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