Increase In Interstitial Collagen Research Articles

P163 EMPAGLIFLOZIN AND ITS CARDIOPROTECTIVE EFFECTS IN THE HEART DURING VOLUME OVERLOAD IN AN EXPERIMENTAL MODEL INDUCED BY AORTOCAVAL FISTULA

Mitral regurgitation causes volume overload of the heart, and due to the deregulation of extracellular matrix proteins, the electrical communication between cardiomyocytes provided by connexin channels is disturbed. The aim of this study was to investigate the effect of cardiac volume overload (VO) on its functional parameters, structural remodeling, and connexin43 (Cx43), a protein ensuring electrical synchronization of the heart, and the cardioprotective effect of empagliflozin. Cardiac VO was induced by surgical aortocaval fistula (ACF) in adult, 6-month-old male Wistar rats. Mitral regurgitation and VO developed within four weeks. Subsequently, empagliflozin was administered daily for five weeks at a dose of 10 mg/kg of body weight. Echocardiographic examination showed increased cardiac output and increased Vmax of the aortic valve, decreased ejection fraction and fractional shortening due to ACF-VO. Echocardiographic parameters showed an increase in the diameter of the left ventricle in both systole and diastole, as well as an increase in the volume of the left ventricle at the end of systole and diastole. Empagliflozin normalized the negative parameters in the ACF-VO group. Myocardial protein Cx43 levels were reduced by ACF-VO but normalized by empagliflozin treatment. In addition, empagliflozin suppressed the ACF-VO-induced increase in interstitial collagen detected by van Gieson staining. These findings were consistent with increased protein expression of MMP-2, which is involved in structural remodeling of the extracellular matrix, and was normalized by empagliflozin. Protein kinase C epsilon levels associated with Cx43 and extracellular matrix modulation were reduced by ACF-VO and normalized by empagliflozin. However, protein levels of pro-apoptotic and pro-hypertrophic protein kinase C delta were increased by ACF-VO and normalized by empagliflozin. Protein expression of transforming growth factor beta as well as fibroblast growth factor 21 was increased by ACF-VO and normalized by empagliflozin. The results indicate a cardioprotective effect of empagliflozin due to suppression of myocardial structural remodeling and preservation of Cx43 in an experimental model simulating volume overload. The research was supported by grants APVV-21-0410,VEGA-2/0006/23,VEGA-2/0133/24,VEGA-2/0002/20

Abstract Tu097: Titin cleavage induces loss of myocardial tensional homeostasis driving a rapid TGF-beta-independent fibrotic response

Background: Cardiomyocytes (CMs) are subjected to different types of mechanical loads in the myocardium. Depending on its origin these loads can be classified as extracellular (extracellular matrix-ECM towards CMs), intracellular (exerted by the CM contraction machinery, i.e. the sarcomeres) or intercellular (exerted between CMs). In the myocardium, all these loads are actively maintained in tensional homeostasis (TH). In this context, the study of the sarcomere protein titin is essential, because its I-band region is considered as a hub for mechanotransduction, mostly of intracellular mechanical cues. Research Questions: It has been shown that mechanical disruption of the ECM affects the myocardial TH, leading to morphological changes inside CMs. However, potential alteration of TH resulting from defective mechanics of CM proteins remains unknown due to the lack of specific tools for in vivo studies. Aims: Here we explore the in vivo effects on myocardial TH of an abrupt tensional unloading of titin. Methods: We have experimentally challenged CMs to a tensional unloading by means of titin cleavage in vivo using TEVs-TTN mice. In this model, a cassette with a Tobacco Etch Virus protease (TEVp) recognition sequence has been included in the I-band of titin. Transducing TEVp with adeno-associated virus (AAV), we were able to cleave titin in vivo , ceasing only the mechanical properties of the protein. Samples were analyzed using an array of techniques including histology, immunofluorescence, and bulk and single nuclei RNA sequencing. Results: Our results show that a mosaic expression of TEVp, which does not affect cell viability, cleaving no more than 30% of total titin. However, this leads to a fast fibrotic response (less than six days), characterized by an increase in interstitial collagen and a response from cardiac fibroblasts population. Transcriptional and phospo-SMAD2/3 analysis results suggest that this ECM response occurs without noticeable activation of the TGFβ canonical pathway. Conclusion: Taken together, our results show experimentally for the first time that titin is not only a mechanotransducer of intracellular cues. In fact, mechanical abrogation of the I-band leads to a fast ECM response mediated by cardiac fibroblasts.

Titin mechanical unloading disrupts sarcomere tensional homeostasis triggering fast, non-canonical myocardial fibrosis

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): We acknowledge funding from Ministerio de Ciencia e Innovación (Madrid, Spain) through grant FJC2021 047055-I and European Research Council (ERC) through grant H2020-ERC-2020-COG (ProtMechanics-Live). Background/Introduction Stresses exerted during myocardial contraction and relaxation cycles are mainly braced by mechanical proteins located in the extracellular matrix (ECM) and cardiomyocytes (CMs). Titin (TTN) is a gigantic protein that scaffolds the sarcomere (i.e. the basic unit of contraction). Since most of the CM cytoskeleton is occupied by sarcomeres, titin is an essential structural hub for the maintenance of a homeostatic level of mechanical tension. Purpose It has been shown that mechanical disruption of the ECM affects the equilibrium forces between CMs and the ECM, leading to morphological changes in sarcomeres. However, potential alteration of tensional homeostasis resulting from defective mechanics of CM proteins remains unknown due to the lack of tools for in vivo studies. Therefore, the aim of this study is to explore the in vivo response of the myocardium to the abrupt cessation of the mechanical properties of titin. Methods For this purpose, we have experimentally challenged CMs to a tensional unloading by means of a titin cleavage model (TEVs-TTN) [1], both in vitro, using neonatal CM (NMCM), and in vivo. In this model, a cassette with a tobacco etch virus protease (TEVp) recognition sequence has been included in the I-band of titin. Transducing TEVp with adeno-associated virus (myoAAV), we were able to cleave titin in living CMs, ceasing only its mechanical properties. Samples were analyzed using an array of techniques such as histology, immunofluorescence and bulk and single-nucleus RNAseq. Results Our results show that while in NMCM we obtain a complete titin cleavage, in vivo we observe mosaic expression of TEVp and no more than 30% cleavage of the protein. In both cases, TEVp expression in TEVs-TTN does not affect cell viability. However, mild myocardial titin cleavage leads to a fibrotic response in less than six days, characterized by an increase in interstitial collagen and an expansion of cardiac fibroblasts population. Transcriptional and phospo-SMAD2/3 analysis results suggest that this fibrotic response occurs without activation of the canonical TGFβ pathway. Conclusion Taken together, our results suggest that the loss of titin mechanical-structural function in cardiomyocytes derived from the cleavage of a single peptide bond elicits a fast fibrotic compensatory response in the myocardium through intercellular communication with fibroblasts.

Renal Microvascular Rarefaction Accompanies Interstitial Fibrosis and Tubular Damage in One Kidney‐Deoxycorticosterone Acetate‐Salt (1K/DOCA/salt)‐Dependent Hypertension

High dietary salt intake is a risk factor for hypertension and chronic kidney disease. However, the mechanisms involved remain poorly understood. Microvascular rarefaction ‐ defined as the loss of arterioles and capillaries within a tissue ‐ has the potential to increase total peripheral vascular resistance and cause local hypoxia and tissue damage. Here we utilised one kidney‐deoxycorticosterone acetate‐salt (1K/DOCA/salt)‐treated mice to investigate whether the development of hypertension and renal damage are associated with microvascular rarefaction. C57BL6/J mice were uninephrectomised and treated for 21 d with DOCA (2.4 mg/day, s.c.) and 0.9% NaCl in the drinking water. Normotensive controls were uninephrectomized and received a placebo pellet and normal drinking water (1K/placebo). Blood pressure (BP) was measured via tail cuff; renal vascular density was assessed immunohistochemically as the percentage area of CD31‐positive staining; renal collagen was detected with picrosirius red; and hematoxylin and eosin were used to visualise renal inflammation and histopathology. 1K/DOCA/salt‐treated mice had higher BPs (160±4 vs. 126±4 mmHg; n≥7; P≤0.0001) and renal weights (321±16 vs. 251±13 mg; n≥7; P<0.05) than 1K/placebo mice. 1K/DOCA/salt‐treated mice also displayed marked renal fibrosis, inflammation and damage as evidenced by a 10‐fold increase in interstitial collagen (n≥7; P<0.05), the presence of inflammatory cells, tubular dilatation and atrophy, and a loss of epithelial brush borders. Importantly, 1K/DOCA/salt‐treatment was associated with a 50% reduction in microvascular density (n≥7; P<0.05), mainly due to a loss of peritubular capillaries. Correlation analyses revealed a trend for an inverse association of peritubular capillary density with collagen deposition (n≥7; r= −0.7058; P= 0.117), but not with BP or inflammatory cell infiltration. Low magnification analysis of kidney sections revealed that peritubular capillary loss occurred in bands emanating from the arcuate arteries. Moreover, collagen deposition, inflammatory cell infiltration and tubular atrophy displayed a similar banding pattern. In summary, these studies reveal a likely association between microvascular rarefaction and fibrosis during the development of 1K/DOCA/salt‐dependent hypertension and renal damage. Future studies will assess whether strategies aimed at maintaining renal microvascular density can minimise renal fibrosis and damage.Support or Funding InformationThe work was funded by the National Health and Medical Research Council of Australia (GNT1143674)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cinnabar induces renal inflammation and fibrogenesis in rats.

The purpose of this study was to investigate whether cinnabar causes renal inflammation and fibrosis in rats. Rats were dosed orally with cinnabar (1 g/kg/day) for 8 weeks or 12 weeks. The control rats were treated with solvent (5% carboxymethylcellulose solution) over the same time periods, respectively. Renal mercury (RHg), urinary mercury (UHg), serum creatinine (SCr), urine kidney injury molecule 1 (KIM-1), renal pathology, and renal mediators were examined. At both 8 weeks and 12 weeks, RHg, UHg, and urine KIM-1 were significantly higher in the cinnabar group than in the control group, although SCr was unchanged. Kidney lesions in the cinnabar-treated rats occurred mainly in the tubules and interstitium, including vacuolization, protein casts, infiltration of inflammatory cells, and slight increase in interstitial collagen. In addition, mild mesangial proliferation was observed in glomeruli. Moreover, the expression of inflammatory and fibrogenic mediators was upregulated in the cinnabar group. In conclusion, cinnabar may cause kidney damage due to the accumulation of mercury, and renal inflammation and slight fibrogenesis may occur in rats. In the clinic, the potential risk of renal injury due to the prolonged consumption of cinnabar should be considered even though the agent is relatively nontoxic.

Ranolazine protects from doxorubicin‐induced oxidative stress and cardiac dysfunction

Doxorubicin is widely used against cancer; however, it can produce heart failure (HF). Among other hallmarks, oxidative stress is a major contributor to HF pathophysiology. The late INa inhibitor ranolazine has proven effective in treating experimental HF. Since elevated [Na+]i is present in failing myocytes, and has been recently linked with reactive oxygen species (ROS) production, our aim was to assess whether ranolazine prevents doxorubicin-induced cardiotoxicity, and whether blunted oxidative stress is a mechanism accounting for such protection. In C57BL6 mice, doxorubicin treatment for 7 days produced LV dilation and decreased echo-measured fractional shortening (FS). Ranolazine (305 mg/kg/day) prevented LV dilation and dysfunction when co-administered with doxorubicin. Doxorubicin-induced cardiotoxicity was accompanied instead by elevations in atrial natriuretic peptide (ANP), BNP, connective tissue growth factor (CTGF), and matrix metalloproteinase 2 (MMP2) mRNAs, which were not elevated on co-treatment with ranolazine. Alterations in extracellular matrix remodelling were confirmed by an increase in interstitial collagen, which did not rise in ranolazine-co-treated hearts. Levels of poly(ADP-ribose) polymerase (PARP) and pro-caspase-3 measured by western blotting were lowered with doxorubicin, with increased cleavage of caspase-3, indicating activation of the proapoptotic machinery. Again, ranolazine prevented this activation. Furthermore, in HL-1 cardiomyocytes transfected with HyPer to monitor H2O2 emission, besides reducing the extent of cell death, ranolazine prevented the occurrence of oxidative stress caused by doxorubicin. Interestingly, similar protective results were obtained with the Na+/Ca2+ exchanger (NCX) inhibitor KB-R7943. Ranolazine protects against experimental doxorubicin cardiotoxicity. Such protection is accompanied by a reduction in oxidative stress, suggesting that INa modulates cardiac redox balance, resulting in functional and morphological derangements.

1682P - Anthracycline-Induced Cardiotoxicity in Mice is Prevented by Late INa Inhibition with Ranolazine, With Improvement in Heart Function, Fibrosis and Apoptosis

ABSTRACT Background Doxorubicin (DOX) produces a well-known cardiomyopathy through multiple mechanisms, which include, among many, Ca2+ overload due to reduced SERCA2a activity and inappropriate opening of the RyR2, and impaired myocardial energetics. DOX generates Reactive Oxygen and Nitrogen Species (ROS and RNS), posing the heart at increased demand for oxygen, setting the stage for metabolic ischemia that also activates late INa, target of ranolazine (RAN). Here, we aim at assessing whether RAN, diminishing intracellular Ca2+ through inhibition of late INa, and enhancing myocardial glucose utilization (and/or reverting impairment of glucose utilization caused by chemotherapy) prevents DOX cardiotoxicity. Methods We measured left ventricular (LV) function with fractional shortening (FS) by echocardiography in C57BL6 mice, 2-4 mo old, pretreated with RAN (370mg/kg/day, a dose comparable to the one used in humans) per os for 3 days. RAN was then administered for additional 7 days, alone and together with DOX (2.17mg/kg/day ip), according to our well established protocol. Hearts were then excised, mRNA expression was analyzed by qRT- PCR, interstitial fibrosis with picrosirius red staining. By Western blotting, we measured the activation of the apoptotic pathway. Results After 7 days with DOX, FS decreased to 50 ± 2%, p = .002 vs 60 ± 1% (sham). RAN alone did not change FS (59 ± 2%). Interestingly, in mice treated with RAN + DOX, the reduction in FS was milder: 57 ± 1%, p = 0.01 vs DOX alone. DOX-cardiotoxicity was accompanied by significant elevations in ANP (1000 folds), BNP (500 folds), CTGF (26 folds) and MMP2 (81 folds) mRNAs, while co-treatment with RAN significantly lowered these same genes compared to DOX. The alterations in extracellular matrix remodeling were confirmed by an increase of interstitial collagen with DOX (3.66%), p = .004 vs 2.19% (sham), which was normal in hearts co-treated with RAN (2.02%, p = .0002 vs DOX). Finally, the levels of PARP and pro-Caspase 3 were significantly decreased in DOX (indicating activation of apoptosis) but not in RAN + DOX. Conclusions In mice, RAN prevents DOX cardiotoxic effects. We plan to test RAN as a cardioprotective agent with other antineoplastic cardiotoxic drugs in mice, and to better characterize the cardioprotective mechanisms of RAN in these settings. Disclosure All authors have declared no conflicts of interest.

Abstract 313: Interleukin-6 Mediates Concentric Hypertrophy, Diastolic Dysfunction and Myocardial Fibrosis in Rats

Induction of inflammatory cytokines has been implicated in the progression of myocardial remodeling and heart failure (HF). Increased levels of circulating TNF-α and interleukin-6 (IL-6) in patients with HF, suggest that these cytokines may be involved in the pathogenesis of heart disease. In previous studies we have shown that TNF-α is an important contributor to the adverse myocardial remodeling. TNF-α is known to mediate collagen degradation as well as in-series sarcomeric addition contributing to ventricular dilatation. However, the effects of IL-6 on cardiac remodeling in vivo have not been investigated. Accordingly, in this study we explore the hypothesis that up-regulation of IL-6 mediates adverse myocardial remodeling. To this end, a group of adult male Sprague Dawley rats was infused with IL-6 (2.5 μg/kg/hr, IP) for 7 days via osmotic minipump and compared to aged-matched shams. LV pressure, size, and function were measured using a blood-perfused isolated heart preparation. At the end of the experiment, hearts were weighed and analyzed for collagen volume fraction (CVF) and isolated cardiomyocyte size. The EDP-EDV (End Diastolic Pressure and Volume) relationship provided that IL-6 infusion produced LV stiffness and a clear tendency for a shift of the EDP-EDV curve to the left due to ventricular hypertrophy and diastolic dysfunction. LV weight differences demonstrate concentric hypertrophy (749 mg versus 660 mg in control hearts; p< 0.05) and a marked increase in interstitial collagen in the IL-6 infused hearts relative to that in control hearts (CVF of 6.2% vs. 1.7%, respectively; p< 0.001). The cardiomyocyte hypertrophy at the cellular level also reflected a concentric phenotype, with cells being significantly longer and thicker (18% and 32%, respectively; p< 0.01). These novel observations demonstrate a direct effect of IL-6 on cardiac remodeling in vivo, which in contrast to TNF-α, induces a dramatic myocardial fibrosis together with concentric cardiac hypertrophy. This suggests that IL-6 may contribute to the development of diastolic dysfunction, and as such could drive the transition to heart failure.

Systemic Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Delivery Shows Antiatherosclerotic Activity in Apolipoprotein E–Null Diabetic Mice

Although in vitro studies have suggested that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) might be involved in vascular biology, its potential role in the pathogenesis and/or treatment of atherosclerosis has not been investigated. Both recombinant human TRAIL and an adeno-associated virus vector expressing human TRAIL were used to deliver TRAIL in apolipoprotein E (apoE)-null mice in which diabetes mellitus was induced by destruction of islet cells with streptozotocin. Diabetes in apoE-null mice was associated with a significant increase in atherosclerotic plaque area and complexity in the aorta as assessed by a marked increase in interstitial collagen, cellular proliferation, and macrophage infiltration and a focal loss of endothelial coverage. Repeated intraperitoneal injections of recombinant human TRAIL and a single intravenous injection of adeno-associated virus-human TRAIL significantly attenuated the development of atherosclerotic plaques in apoE-null animals. TRAIL also markedly affected the cellular composition of plaque lesions by inducing apoptosis of infiltrating macrophages and increasing the vascular smooth muscle cell content. Moreover, TRAIL promoted the in vitro migration of cultured human aortic vascular smooth muscle cells but not of monocytes or macrophages. Conversely, TRAIL selectively induced apoptosis of human cultured macrophages but not of vascular smooth muscle cells. Overall, data from the present study indicate that atherosclerosis in diabetic apoE-null mice is ameliorated by systemic TRAIL administration and that adeno-associated virus-mediated TRAIL gene delivery might represent an innovative method for the therapy of diabetic vascular diseases.

The Relaxin Gene‐Knockout Mouse: A Model of Progressive Fibrosis

Relaxin is well known for its actions on collagen remodeling. To improve our understanding of the physiologic role(s) of relaxin, the relaxin gene-knockout (RLX-KO) mouse was established by our group and subsequently phenotyped. Pregnant RLX-KO mice underwent inadequate development of the pubic symphysis as well as the mammary glands and nipples compared to wild-type mice, thus preventing lactation. Later studies showed that these deficiencies were associated with increased collagen, primarily in the nipple and vagina. Analysis of male RLX-KO mice also demonstrated inadequate reproductive tract development. The testis, epididymis, and prostate of RLX-KO mice showed delayed tissue maturation and growth associated with increased collagen deposition. In nonreproductive tissues, an age-related increase in interstitial collagen (fibrosis) was also detected in the lung, heart, and kidneys of RLX-KO mice and was associated with organ dysfunction. From 6-9 months of age and onwards, all organs of RLX-KO mice, particularly male mice, underwent progressive increases in tissue weight and collagen content (all P < .05) compared with wild-type animals. The increased fibrosis contributed to bronchiole epithelium thickening and alveolar congestion (lung), atrial hypertrophy and increased ventricular chamber stiffness (heart) in addition to glomerulosclerosis (kidney). Treatment of RLX-KO mice with recombinant human relaxin in early and developed stages of fibrosis caused the reversal of collagen deposition in the lung, heart, and kidneys. Together, these findings suggest that relaxin is a naturally occurring inhibitor of collagen deposition during normal development, aging, and pregnancy and can be used to prevent the progression of fibrosis.

Interaction between isolated human myocardial mast cells and cultured fibroblasts 1

Introduction Previously, we reported an increase in interstitial collagen and total mast-cell numbers in heart failure versus normal myocardium. A secondary increase, primarily in chymase-negative mast cells, occurred following LVAD support compared to matched pre-LVAD tissue samples and was associated with a decrease in interstitial collagen and bFGF. To further elucidate the changes in interstitial collagen, we investigated the direct interaction between mast cells, isolated from failing myocardium with or without previous LVAD support, and human fibroblasts in a coculture model. Additionally, the expression of HSP-47, the pro-collagen-specific chaperone protein, was determined in the particular myocardium. Materials and methods Myocardial tissue was obtained from 10 patients with end-stage dilated cardiomyopathy (DCM) at the time of transplantation. Five patients were transplanted following LVAD support, five patients without previous LVAD support. Mast cells were isolated according to a standard protocol, including collagenase digestion and cell separation. The isolated mast cells were co-cultured with human fibroblasts for 12 h, with or without stimulation of degranulation, and protein synthesis was measured by [ 3H]-proline incorporation. HSP-47 immunostaining was performed in the different myocardial samples and the positive cells were quantified. Results Stimulated mast cells isolated from DCM tissue (without previous LVAD support) caused a 92% increase in [ 3H]-proline incorporation and consequently in protein production in fibroblasts compared to mast-cell free culture ( P < 0.01), while conversely stimulated mast cells isolated from LVAD supported myocardium decreased the [ 3H]-proline incorporation by 63% ( P < 0.01) below baseline. Nonstimulated mast cells did not significantly alter the protein production over baseline. There was also a significant increase in the number of HSP-47-positive cells in DCM myocardium compared to normal ( P < 0.01) and there was a shift toward normal after LVAD support ( P < 0.01). Conclusion We demonstrate that fibroblast protein production in vitro is significantly altered by mast cells and that the direction of change is dependent on whether myocardium was supported by LVAD. We suggest that under long-term LVAD support there is a phenotypic alteration in myocardial mast cells, which leads to a change in concentration and/or composition of mediators, capable of re-remodeling the myocardial matrix.

Interaction between isolated human myocardial mast cells and cultured fibroblasts

Introduction: Previously, we reported an increase in interstitial collagen and total mast cell numbers in heart failure versus normal myocardium. A secondary increase, primarily in chymase-negative mast cells, occurred following LVAD-support compared to matched pre-LVAD tissue samples and was associated with a decrease in interstitial collagen and bFGF. We investigated the direct interaction between mast cells, isolated from failing myocardium with or without previous LVAD support, and human fibroblasts in a co-culture model. Methods: Myocardial tissue was obtained from 10 patients with endstage dilated cardiomyopathy (DCM) at the time of transplantation. Five patients were transplanted following LVAD support, five patients without previous LVAD support. Mast cells were isolated according to a standard protocol, including collagenase digestion and cell separation. The isolated mast cells were co-cultured with human fibroblasts for 12 hours, with or without stimulation of degranulation and protein synthesis was measured by [3H]-proline incorporation. Results: Mast cells isolated from unsupported DCM tissue caused a 92% increase in [3H]-proline incorporation in fibroblasts after stimulation compared to mast cell free culture (p < 0.01), while mast cells isolated from LVAD supported myocardium decreased the [3H]-proline incorporation by 63% (p < 0.01) and consequently the protein production. Unstimulated mast cells did not significantly alter the protein production over baseline. Conclusion: We demonstrate that fibroblast protein production in vitro is significantly altered by mast cells and that the direction of change is dependent on whether myocardium was supported by LVAD. We suggest, that under long-term LVAD support there is a phenotypic alteration in mast cells, which leads to a change in mediators’ concentration and/or composition, capable of re-remodeling the myocardial matrix.

Ventricular-specific expression of angiotensin II type 2 receptors causes dilated cardiomyopathy and heart failure in transgenic mice.

The angiotensin II type 2 (AT2) receptor is upregulated in the left ventricle in heart failure, but its pathophysiological roles in vivo are not understood. In the present study, AT2 receptors were expressed in transgenic (TG) mice using the ventricular-specific myosin light-chain (MLC-2v) promoter. In TG compared with nontransgenic (NTG) mice, in vivo left ventricular (LV) systolic pressure and peak +dP/dt were depressed while LV diastolic pressure was elevated (P < 0.05). Echocardiography showed severely depressed LV fractional shortening, increased systolic and diastolic dimensions, and wall thinning (P < 0.05). Confocal and electron microscopy studies revealed an increase in the size of myocytes and interstitial spaces as well as an increase in interstitial collagen, disruption of the Z-band, and changes in cytochrome c localization. The changes were most prominent in the highest-expressing TG line, which implies a dose-response relationship. AT2 overexpression was also directly associated with the increase of phosphorylated protein levels of PKC-alpha, PKC-beta, and p70S6 kinase. These data demonstrate that ventricular myocyte-specific expression of AT2 receptors promotes the development of dilated cardiomyopathy and heart failure in vivo.

Microvascular and tubulointerstitial injury associated with chronic hypoxia-induced hypertension

Rats submitted to chronic hypoxia develop hypertension that persists despite cessation of the hypoxia or correction of the hematocrit. We examined whether chronic hypoxia might induce subtle renal injury since studies in other animal models of hypertension suggest this may cause persistent hypertension. Chronic hypoxia was induced in rats by placement in a hypobaric chamber for up to 24 days. Blood pressure and kidney biopsies were performed at baseline, 6 hours, 24 hours, and 24 days of hypoxia. Chronic hypoxia induced hypertension and erythrocytosis at 24 days. Acute hypoxia was associated with endothelial cell swelling in arterioles (6 and 24 hours), followed by thickening of the arterioles at 24 days. Subtle tubulointerstitial injury and inflammation occurred and was progressive. The influx of macrophages increased steadily over the 24 days and was associated with a progressive increase in interstitial collagen III deposition. Hypoxia was associated with increased tubular expression of osteopontin as early as 6 hours, the same period when an increase of proximal tubular cell proliferation occurred. Interstitial cell proliferation peaked twice, at 6 hours and at 24 days. Glomerular hypertrophy was manifest at 24 days. Both afferent arteriolar disease and subtle tubulointerstitial inflammation and injury occur early in hypoxic rats. These changes may predispose these animals to persistent hypertension.

Increase of Interstitial Collagen in the Mouse Endometrium During Decidualization

Increase of Interstitial Collagen in the Mouse Endometrium During Decidualization

Increase of Interstitial Collagen in the Mouse Endometrium During Decidualization

Decidualization in the mouse consists of an extensive remodeling of the endometrial extracellular matrix, resulting in a reduction of the extracellular spaces, an increase in the diameter of collagen fibrils, and changes in the relative ratio of different types of glycosaminoglycans. To assess the dynamic changes of the endometrial extracellular matrix during decidualization, collagen was analyzed biochemically and immunochemically in the endometrium of nulliparous and day 5 to day 8 pregnant mice. The amount of collagen per gram dry weight was higher in the endometrium of implantation sites than in interimplantation sites. Collagen types I, III, and V were the main components of the endometrium of nulliparous and pregnant animals. The amount of collagen type V was higher in the endometrium of pregnant animals than in nulliparous ones. A relative unusual homotrimeric form of collagen type V, probably formed by [ f 1(V)] 3 , was detected in pregnant endometrium by gel eletrophoresis and immunoblotting.

Lack of influence of cyclosporin A on levels of gingival procollagen types I and III mRNAs in rats of different ages

Previous studies showed that gingival overgrowth following cyclosporin A (CsA) administration is not associated with an increase in interstitial collagen. It also was shown that CsA causes a significant decrease in collagen content within the gingival stroma. In order to determine whether this decrease is caused by down-regulation of collagen mRNA, the procollagen mRNA level in gingiva of young and old rats was measured and correlated with the ratio of interstitial collagen to DNA in these regions. Hybridization of 32P-labelled cDNA probes for procollagen types I and III with total RNA extracted from the molar gingiva showed that administration of CsA did not change the steady-state levels of mRNAs for both procollagens in the gingiva of either young or old rats. The ratio of gingival interstitial collagen to DNA was significantly reduced in the CsA-treated animals (4.2 ± 0.85) relative to the controls (7.8 ± 1.6). It is concluded that the reduction in interstitial collagen following CsA treatment is not age-related, and is most probably caused by increased degradation rather by decreased biosynthesis.

Remodelling of intramyocardial arterioles and extracellular matrix in patients with arterial hypertension and impaired coronary reserve.

The heart in pressure overload is threatened by the development of diastolic and systolic dysfunction even in the absence of coronary heart disease. In pressure overload, systolic wall stress leads to an increase in left ventricular mass through hypertrophy of myocytes. An activation of myocytic as well as non-myocytic cells is present. An increase in interstitial collagen accompanies hypertrophy of the myocytes. Thus, myocytic hypertrophy and fibrosis cause diastolic dysfunction early on, even when systolic function is still well preserved. In hypertensive heart disease, the coronary microcirculation is remodelled by thickening of the walls of intramyocardial arterioles in relation to their lumen and by an increase in periarteriolar fibrosis. This remodelling of the intramyocardial vasculature is combined with a reduction in coronary vasodilator reserve that may lead to malnutrition and malperfusion of the hypertrophied myocytes. The combined processes of myocytic hypertrophy, vascular remodelling and increased fibrosis may be important in the process of ventricular dilatation and failure in hypertensive heart disease.

Cyclosporin A: recent developments in the mechanism of action and clinical application

Previous studies showed that gingival overgrowth following cyclosporin A (CsA) administration is not associated with an increase in interstitial collagen. It also was shown that CsA causes a significant decrease in collagen content within the gingival stroma. In order to determine whether this decrease is caused by down-regulation of collagen mRNA, the procollagen mRNA level in gingiva of young and old rats was measured and correlated with the ratio of interstitial collagen to DNA in these regions. Hybridization of 32P-labelled cDNA probes for procollagen types I and III with total RNA extracted from the molar gingiva showed that administration of CsA did not change the steady-state levels of mRNAs for both procollagens in the gingiva of either young or old rats. The ratio of gingival interstitial collagen to DNA was significantly reduced in the CsA-treated animals (4.2 ± 0.85) relative to the controls (7.8 ± 1.6). It is concluded that the reduction in interstitial collagen following CsA treatment is not age-related, and is most probably caused by increased degradation rather by decreased biosynthesis.

Analgesic nephropathy: A clinicopathologic study using electron microscopy

A Clinicopathologic study was made in twenty-six patients who had consumed at least 2 kg of both acetylsalicylic acid and phenacetin. Renal function varied from normal to end-stage. Conventional histologic and electron microscopic studies confirmed a medullary site for the earliest lesion, which appeared to be an increase in interstitial collagen. Other early abnormalities included focal thickening of tubular basement membranes, degeneration, atrophy or loss of tubular epithelium, and casts. These abnormalities were apparent when renal function was not demonstrably reduced. They progressed to become a diffuse sclerosis or necrosis of the medulla, and only then were cortical histologic abnormalities present. These included peritubular, interstitial and periglomerular fibrosis; tubular atrophy, dilatation and basement membrane thickening; and in some focal round cell infiltration. Results of immunofluorescent microscopy were negative for immunoglobulin G (IgG) and beta 1-C globulin ( β 1 C) in kidney tissue from six patients. The earliest functional lesion was impaired ability to concentrate urine. When creatinine clearance was even slightly reduced, there was medullary sclerosis and in some patients pyelographic evidence of extensive papillary necrosis. Bacteriuria was found mainly in patients whose renal function was considerably reduced, and pyelonephritis, when present, appeared to be secondary. Renal function stabilized or improved in most patients who stopped taking mixed analgesics, but deteriorated further in those who did not.