Interstitial Collagen Content Research Articles

The impact of moderate endurance exercise on cardiac telomeres and cardiovascular remodeling in obese rats.

Hypercaloric nutrition and physical inactivity cause obesity, a potential driver of myocardial apoptosis and senescence that may accelerate cardiac aging. Although physical activity reduces mortality, its impact on myocardial aging is insufficiently understood. Here we investigated the effects of a hypercaloric high-fat diet (HFD) and regular exercise training on cardiac cells telomeres and histomorphometric indices of cardiac aging. Ninety-six 4-months old female Sprague-Dawley rats were fed for 10 months normal (ND) or a HFD diet. Half of the animals in each group performed 30 min treadmill-running sessions on 5 consecutive days per week. At study end, cardiomyocyte cross-sectional area (CSA), interstitial collagen content, vascular density, apoptotic and senescent cells, relative telomere length (RTL), and expression of telomerase-reverse transcriptase (Tert) as marker of telomere-related senescence and apoptosis were analyzed. Compared to ND, the HFD group developed obesity, higher CSA, lower capillary density and tended to have more apoptotic cardiomyocytes and interstitials cells. Myocardial RTL and the expression of Terf-1 and Terf-2 were comparable in sedentary HFD and ND animals. In the HFD group, regular moderate endurance exercise improved myocardial vascularization, but had no effect on CSA or apoptosis. Notably, the combination of exercise and HFD increased senescence when compared to sedentary ND or HFD, and reduced RTL when compared to exercise ND animals. Exercising HFD animals also showed a trend toward higher Tert expression compared to all other groups. In addition, exercise reduced Terf-1 expression regardless of diet. HFD-induced obesity showed no effects on myocardial telomeres and induced only mild morphologic alterations. Summarized, long-term moderate endurance exercise partially reverses HFD-induced effects but may even trigger cardiac remodeling in the context of obesity.

Abstract 15337: Chronic Oral Finerenone Treatment Does Not Improve Left Ventricular Diastolic Compliance in Swine With Repetitive Pressure Overload-Induced HFpEF

Introduction: The non-steroidal mineralocorticoid receptor antagonist finerenone has emerged as a potential treatment for heart failure with preserved ejection fraction (HFpEF) based, in part, on initial evidence of its anti-fibrotic properties. However, it is unclear whether chronic oral finerenone therapy can reverse established cardiac fibrosis. Accordingly, we tested the efficacy of chronic oral finerenone in a porcine model of HFpEF characterized by interstitial fibrosis and reduced left ventricular (LV) diastolic compliance in the absence of co-morbidities or anatomic LV hypertrophy. Methods: Swine (n=12) were subjected to daily (1 hr) episodes of repetitive LV pressure overload (RPO) for 2 weeks using phenylephrine (24 mg/hr) to transiently elevate LVEDP to ~30 mm Hg. After 2 weeks, RPO was discontinued and animals were followed for an additional 4 weeks, during which a subset received oral finerenone (20 mg/day; n=6). LV end-diastolic volume (EDV) and pressure (EDP) were measured at normal and elevated preload to assess LV compliance (ΔLVEDV/ΔLVEDP) and post-mortem picrosirius red staining was performed to quantify interstitial fibrosis vs. normal controls (n=8). Results: Four-weeks after cessation of RPO, untreated animals exhibited a significant increase in interstitial fibrosis vs. normal controls that was attenuated in finerenone-treated animals ( Figure A ). Despite this regression of interstitial collagen, a persistent reduction in LV diastolic compliance was observed after finerenone treatment (1.18 ± 0.33 mL/mmHg vs. 2.11 ± 0.22 mL/mmHg in normal controls; p=0.04) that was comparable to that of untreated animals after RPO (0.97 ± 0.22 mL/mmHg; p=0.61; Figure B ). Conclusion: Four weeks of chronic oral finerenone treatment does not restore LV diastolic compliance in swine with RPO-induced HFpEF despite regression of interstitial collagen content, suggesting that other factors may contribute to diastolic stiffening in this model.

Only Acute but Not Chronic Thrombocytopenia Protects Mice against Left Ventricular Dysfunction after Acute Myocardial Infarction.

Platelets are major players of thrombosis and inflammation after acute myocardial infarction (AMI). The impact of thrombocytopenia on platelet-induced cellular processes post AMI is not well defined. The left anterior descending artery was ligated in C57/Bl6 mice and in two thrombocytopenic mouse models to induce AMI. Platelets from STEMI patients and from C57/Bl6 mice displayed enhanced platelet activation after AMI. This allows platelets to migrate into the infarct but not into the remote zone of the left ventricle. Acute thrombocytopenia by antibody-induced platelet depletion resulted in reduced infarct size and improved cardiac function 24 h and 21 days post AMI. This was due to reduced platelet-mediated inflammation after 24 h and reduced scar formation after 21 days post AMI. The collagen composition and interstitial collagen content in the left ventricle were altered due to platelet interaction with cardiac fibroblasts. Acute inflammation was also significantly reduced in Mpl-/- mice with chronic thrombocytopenia, but cardiac remodeling was unaltered. Consequently, left ventricular function, infarct size and scar formation in Mpl-/- mice were comparable to controls. This study discovers a novel role for platelets in cardiac remodeling and reveals that acute but not chronic thrombocytopenia protects left ventricular function post AMI.

Repetitive Preload Elevation in Swine with Chronic Coronary Artery Disease Promotes Pro‐Fibrotic Gene Expression and Transmural Fibrosis in Remote Non‐Ischemic Myocardium

ObjectiveUsing implantable telemetry, we recently found that transient elevations in preload are common in swine with multi‐vessel coronary artery disease (CAD), presumably as a result of intermittent demand‐induced regional myocardial ischemia. Based on prior evidence that repetitive preload elevation can lead to interstitial fibrosis and myocardial stiffening in the absence of ischemia, the present study was designed to compare temporal changes in pro‐fibrotic gene expression and transmural variability in interstitial fibrosis in ischemic vs. non‐ischemic areas of the left ventricle (LV) in a porcine model of chronic CAD.MethodsA total of 20 juvenile swine were instrumented with fixed 1.5 mm constrictors on the proximal left anterior descending (LAD) and left circumflex (LCx) coronary arteries and followed for up to 3‐months, during which coronary stenosis severity progressed to >90 % (LAD: 93±2 %; LCx: 91±3 %). Expression of fibronectin (FN1) and lysyl oxidase like‐2 (LOXL‐2) was assessed in ischemic (LAD) and non‐ischemic (RCA) regions of the LV via quantitative PCR 1‐month (n=4) or 3‐months (n=8) after instrumentation and compared to normal controls (n=8). Post‐mortem quantification of interstitial fibrosis was performed on picrosirius red‐stained tissue samples from ischemic and non‐ischemic regions (n=16), with an automated classifier‐based approach to compare regional transmural variability in collagen content to that of normal controls (n=11). Flash‐frozen tissue samples from a sub‐set of animals with CAD (n=4) and normal controls (n=4) were subjected to uniaxial tensile testing to assess passive subendocardial stiffness.ResultsOne month after instrumentation, FN1 expression was 2.5±0.9‐fold higher than control in the ischemic region and 2.7±0.7‐fold higher than control in the non‐ischemic region of the LV (both p<0.05). LOXL2 expression was also significantly higher in the ischemic (2.2 ± 0.6‐fold higher than control) and non‐ischemic (2.4 ± 0.3‐fold higher than control) regions of the LV at this timepoint. Two months later, pro‐fibrotic gene expression had normalized but a significant elevation in interstitial collagen content was observed throughout the LV of animals with CAD (Figure; left panel). Interestingly, subendocardial fibrosis was particularly prominent in the non‐ischemic RCA territory, which coincided with a significant increase in subendocardial passive stiffness in this region (Figure; right panel).ConclusionsIn a porcine model of chronic CAD, repetitive preload elevation is associated with transient changes in pro‐fibrotic gene expression that ultimately lead to transmural fibrosis that is particularly prominent in non‐ischemic areas of the LV. Regional transmural variability in collagen content coincides with changes in the passive stiffness of non‐ischemic myocardium, suggesting that repetitive preload elevation may contribute to fibrosis‐mediated diastolic dysfunction in patients with chronic CAD.

Cardiac up-regulation of NBCe1 emerges as a beneficial consequence of voluntary wheel running in mice

Physical training stimulates the development of physiologic cardiac hypertrophy (CH), being a key event in this process the inhibition of the Na+/H+ exchanger. However, the role of the sodium bicarbonate cotransporter (NBC) has not been explored yet under this circumstance. C57/Bl6 mice were allowed to voluntary exercise (wheel running) for five weeks. Cardiac mass was evaluated by echocardiography and histomorphometry detecting that training promoted the development of physiological CH (heart weight/tibia length ratio, mg/mm: 6.54 ± 0.20 vs 8.81 ± 0.24; interstitial collagen content, %: 3.14 ± 0.63 vs. 1.57 ± 0.27; and cross-sectional area of cardiomyocytes, μm2: 200.6 ± 8.92 vs. 281.9 ± 24.05; sedentary (Sed) and exercised (Ex) mice, respectively). The activity of the electrogenic isoform of the cardiac NBC (NBCe1) was estimated by recording intracellular pH under high potassium concentration and by measuring action potential duration (APD). NBCe1 activity was significantly increased in isolated cardiomyocytes of trained mice. Additionally, the APD was shorter and the alkalization due to high extracellular potassium-induced depolarization was greater in this group, indicating that the NBCe1 was hyperactive. These results are online with the observed myocardial up-regulation of the NBCe1 (Western Blot, %: 100 ± 13.86 vs. 202 ± 29.98; Sed vs. Ex, n = 6 each group). In addition, we detected a reduction in H2O2 production in the myocardium of trained mice.These results support that voluntary training induces the development of physiologic CH with up-regulation of the cardiac NBCe1 in mice. Furthermore, the improvement in the antioxidant capacity contributes to the beneficial cardiovascular consequences of physical training.

Granulocyte colony-stimulating factor attenuates myocardial remodeling and ventricular arrhythmia susceptibility via the JAK2-STAT3 pathway in a rabbit model of coronary microembolization

BackgroundCoronary microembolization (CME) has a poor prognosis, with ventricular arrhythmia being the most serious consequence. Understanding the underlying mechanisms could improve its management. We investigated the effects of granulocyte colony-stimulating factor (G-CSF) on connexin-43 (Cx43) expression and ventricular arrhythmia susceptibility after CME.MethodsForty male rabbits were randomized into four groups (n = 10 each): Sham, CME, G-CSF, and AG490 (a JAK2 selective inhibitor). Rabbits in the CME, G-CSF, and AG490 groups underwent left anterior descending (LAD) artery catheterization and CME. Animals in the G-CSF and AG490 groups received intraperitoneal injection of G-CSF and G-CSF + AG490, respectively. The ventricular structure was assessed by echocardiography. Ventricular electrical properties were analyzed using cardiac electrophysiology. The myocardial interstitial collagen content and morphologic characteristics were evaluated using Masson and hematoxylin-eosin staining, respectively.ResultsWestern blot and immunohistochemistry were employed to analyze the expressions of Cx43, G-CSF receptor (G-CSFR), JAK2, and STAT3. The ventricular effective refractory period (VERP), VERP dispersion, and inducibility and lethality of ventricular tachycardia/fibrillation were lower in the G-CSF than in the CME group (P < 0.01), indicating less severe myocardial damage and arrhythmias. The G-CSF group showed higher phosphorylated-Cx43 expression (P < 0.01 vs. CME). Those G-CSF-induced changes were reversed by A490, indicating the involvement of JAK2. G-CSFR, phosphorylated-JAK2, and phosphorylated-STAT3 protein levels were higher in the G-CSF group than in the AG490 (P < 0.01) and Sham (P < 0.05) groups.ConclusionG-CSF might attenuate myocardial remodeling via JAK2-STAT3 signaling and thereby reduce ventricular arrhythmia susceptibility after CME.

Cardiac hypertrophy with obesity is augmented after pregnancy in C57BL/6 mice

BackgroundOver a third of reproductive-age women in the USA are obese, and the prevalence of cardiovascular disease (CVD) is rising in premenopausal women. Cardiac hypertrophy is an independent predictor of CVD. In contrast to pregnancy, where transiently increased left ventricular (LV) mass is not associated with cardiac damage, obesity-mediated cardiac hypertrophy is pathological. There is a paucity of data describing the effect of obesity during pregnancy on maternal cardiovascular health. The purpose of this study was to determine the long-term effect of obesity during pregnancy on cardiac function and structure in mice.MethodsFemale C57BL/6 J mice were fed a high-fat (HF) or a low-fat (LF) diet for 20 weeks. After 4 weeks, LF- and HF-fed female mice were either crossed with males to become pregnant or remained non-pregnant controls. Following delivery, pups were euthanized, and females maintained on respective diets. After 20 weeks of diet feeding, cardiac function was quantified by echocardiography, and plasma leptin and adiponectin concentrations quantified in LF- and HF-fed postpartum and nulliparous females. mRNA abundance of genes regulating cardiac hypertrophy and remodeling was quantified from left ventricles using the NanoString nCounter Analysis System. Cardiac fibrosis was assessed from picrosirius red staining of left ventricles.ResultsHF-fed postpartum mice had markedly greater weight gain and fat mass expansion with obesity, associated with significantly increased LV mass, cardiac output, and stroke volume compared with HF-fed nulliparous mice. Plasma leptin, but not adiponectin, concentrations were correlated with LV mass in HF-fed females. HF feeding increased LV posterior wall thickness; however, LV chamber diameter was only increased in HF-fed postpartum females. Despite the marked increase in LV mass in HF-fed postpartum mice, mRNA abundance of genes regulating fibrosis and interstitial collagen content was similar between HF-fed nulliparous and postpartum mice. In contrast, only HF-fed postpartum mice exhibited altered expression of genes regulating the extracellular matrix.ConclusionsThese results suggest that the combined effects of pregnancy and obesity augment cardiac hypertrophy and promote remodeling. The rising prevalence of CVD in premenopausal women may be attributed to an increased prevalence of women entering pregnancy with an overweight or obese BMI.

Abstract P2006: Imbalance Between Collagen Type 1 and 3 in the Remodeling of the Extracellular Matrix Contributes to the Transition to Heart Failure

Background: Hypertension causes cardiac hypertrophy, cardiac dysfunction and heart failure (HF). Despite the importance of cardiac hypertrophy as an independent risk factor for the development of HF, not all hypertrophied hearts will ultimately fail. Alterations in the extracellular matrix have also been shown to contribute to this process. This study aimed to investigate whether alterations in the levels of profibrotic proteins contribute to the transition of compensated cardiac hypertrophy to HF. Methods: Male rats were subjected to abdominal aorta constriction. After 90 days post surgery (dps), the hearts were collected and Western blot was performed to collagens type I and III, MMP-14, MMP-2, TIMP-2, Integrin and TGF-β. The interstitial collagen area was quantified. Cardiac function was evaluated using the Vevo 2100 ultrasound. Data were considered significant when p&lt;0.05. Results: At 90 dps, 75% (30 of 40) presented hypertrophic hearts (HH) and 25% (10 of 40) hypertrophic+dilated hearts (HD) in the echocardiography. In addition, systolic (ejection fraction: 33.5%±1.3; fractional shortening: 21.90±4.3%) and diastolic functions (mitral E-wave velocity: 1279±127 mm/s and E/E' ratio: 68.14±16.78) were importantly reduced in the HD group when compared to HH. Although the interstitial collagen content in the left ventricle was not different between HH (3.63±0.40%) and HD (3.75±0.39%) groups, the amount of type III collagen increased (1.28±0.34 AU) while type I collagen showed significant reduction (0.76±0.16 AU) in the HD group when compared to HH (0.90±0.26 AU and 1.11±0.11 AU, respectively). Important increases were seen in the protein levels of TGF-B, integrin, MMP-14, MMP-2 and TIMP-2 in the HD and HH groups when compared to sham, but there is no difference between HD and HH groups. Conclusion: Although there is significant fibrosis in the hearts of animals after 90 dps, the imbalance between type I and III collagens associated with systolic and diastolic function could contribute to the transition to decompensated hypertrophy in this experimental model of cardiac hypertrophy.

Abstract 528: Restricted Proteasome Heterogeneity Promotes Premature Heart Failure and Deterioration of Associated Protein Levels Upon Continuous β-Adrenergic Stimulation

Introduction: The ubiquitin proteasome-system is a main contributor to cellular proteostasis. The dynamic regulation of proteasome composition and function during cardiac remodeling has been of high interest. To what extend discrete proteasome subunits in turn modify cardiac remodeling, is poorly understood. Objective: Aim of the study was to investigate the contribution of the non-essential proteasome subunit low molecular mass peptide 2 (Lmp2) on cardiac function, tissue and proteome upon induced hypertrophic remodeling. Methods &amp; Results: We found induction of cardiac Lmp2 during isoproterenol (Iso)- and TAC-induced remodeling and hypertrophic cardiomyopathy. Unchallenged adult mice congenitally lacking Lmp2 (KO) were indistinguishable from their WT littermates. After 7 days of continuous Iso administration (30 mg/kg/d) though, cardiac function was significantly reduced in Lmp2 KO mice as compared to WT (fractional shortening: -32% vs. WT, p&lt;0.01, n≥9). Interestingly, reduced cardiac function was accompanied by augmented cardiac remodeling as shown by heart weight to body weight ratio, wall thickness, cardiomyocyte cross-sectional area and interstitial collagen content. Cardiac-restricted re-expression of Lmp2 utilizing AAV9-mediated gene transfer rescued cardiac function and restricted remodeling to WT levels, attributing those findings to cardiac Lmp2. Hence, we analyzed the cardiac proteome utilizing 2-D DIGE. Out of approximately 1500 detected protein spots, 114 were showing significant differences upon cardiac remodeling depending on absolute Lmp2 protein expression (p≤0.05). All spots were manually inspected for correct matching across 10 gels. Subsequently, spots were picked with priority on those with the most prominent changes in abundance and identified by mass spectrometry. For example, we found significantly less carbonic anhydrase 2 and manganese superoxide dismutase in the remodeled myocardium of WT compared to Lmp2 KO mice. Conclusions: Cardiac lack of Lmp2 exacerbates cardiac remodeling, which is reflected in the cardiac proteome by deteriorated levels of proteins with a known pivotal role in human cardiac disease.

Aging and Senescence in Canine Testes.

Senescent cells accumulate with age but tissue-based studies of senescent cells are limited to selected organs from humans, mice, and primates. Cell culture and xenograft studies have indicated that senescent cells in the microenvironment may play a role in tumor proliferation via paracrine activities. Dogs develop age-related conditions, including in the testis, but cellular senescence has not been confirmed. We hypothesized that senescent cells accumulate with age in canine testes and in the microenvironment of testicular tumors. We tested the expression of the established senescence markers γH2AX and p21 on normal formalin-fixed, paraffin-embedded testes from 15 young dogs (<18 months of age) and 15 old dogs (7-15 years of age) and correlated the findings with age-dependent morphological changes. A statistically significant age-dependent increase in the percentage of p21-expressing cells was observed for testicular fibroblasts (4-fold) and Leydig cells (8-fold). However, p21-expressing cells were still a rare event. In contrast, the percentage of γH2AX-positive cells did not increase with age. P21- and γH2AX-expressing cells were rare in the microenvironments of tumors. Age-dependent morphological changes included an increased mean number of Leydig cells per intertubular triangle (2.95-fold) and a decreased spermatogenesis score. To our surprise, no age-related changes were recorded for interstitial collagen content, mean tubular diameter, and epithelial area. Opposed to our expectations based on previous in vitro data, we did not identify evidence of a correlation between age-associated accumulation of senescent cells and testicular tumor development. Understanding the role of the microenvironment in senescence obviously remains a challenging task.

The ACE 2 activator diminazene aceturate (DIZE) improves left ventricular diastolic dysfunction following myocardial infarction in rats

Diminazene aceturate (DIZE) has been reported to enhance the catalytic efficiency of ACE-2 and presumably increases angiotensin 1–7 generation, interfering with cardiac remodeling after myocardial infarction (MI). Our aim was to investigate the chronic effects of DIZE on cardiac dysfunction post-MI. Male Wistar rats underwent myocardial infarction (MI) or SHAM surgery (SO) and were divided into groups treated with DIZE 15 mg/kg/day, s.c. or vehicle (Control). After 4 weeks, the hemodynamic variables were recorded by cardiac catheterism. Hearts were then arrested to obtain the left ventricular (LV) pressure-volume curves in situ. Cardiomyocyte hypertrophy and collagen content were determined by histology. DIZE prevented LV end-diastolic pressure increases in MI rats (MI: 26 ± 3.3 vs. MI-DIZE: 15 ± 1.6 mmHg, P < 0.001) without a significant effect on LV systolic pressure (LVSP). Moreover, DIZE improved LV contractility (+dP/dt, MI: 3014 ± 161 vs. MI-DIZE: 3884 ± 104 mmHg/s, P < 0.001) and relaxation (-dP/dt, MI: −2333 ± 91 vs. MI-DIZE: −2798 ± 120 mmHg/s, P < 0.05). Right ventricular SP was increased in the MI compared to that in the SO group (40 ± 0.6 vs. 30 ± 1.2 mmHg; P < 0.01), and DIZE partially prevented this augmentation. LV stiffness was reduced in MI-DIZE compared with that in MI (0.64 ± 0.01 vs. 0.78 ± 0.02 mmHg/mL; P < 0.01). DIZE treatment reduced the interstitial collagen content by 18% in the surviving LV myocardium. Cardiomyocyte hypertrophy remained unaffected by DIZE treatment. Our findings show that chronic DIZE treatment post-MI attenuates the morphofunctional changes induced by MI in rats. The effects on LV -dP/dt, chamber stiffness and collagen content suggest this drug can be used as a therapeutic agent to reduce interstitial fibrosis and diastolic dysfunction after MI.

A modified elliptical formula to estimate kidney collagen content in a model of chronic kidney disease.

The extent of scarring or renal interstitial collagen deposition in chronic kidney disease (CKD) can only be ascertained by highly invasive, painful and sometimes risky, tissue biopsy. Interestingly, while CKD-related abnormalities in kidney size can often be visualized using ultrasound, not only does the ellipsoid formula used today underestimate true renal size, but the calculated renal size does not inform tubulointerstitial collagen content. We used coronal kidney sections from healthy mice and mice with kidney disease to develop a new formula for estimating renal parenchymal area. While treating the kidney as an ellipse with the major axis (a) the polar distance, this technique involves extending the minor axis (b) into the renal pelvis to obtain a new minor axis, be. The calculated renal parenchymal area is remarkably similar to the true or measured area. Biochemically determined kidney collagen content revealed a strong and positive correlation with the calculated renal parenchymal area. Picrosirius red staining for tubulointerstitial collagen also correlated with calculated renal parenchymal area. The extent of renal scarring, i.e. kidney interstitial collagen content, can now be computed by making just two axial measurements which can easily be accomplished via noninvasive imaging of this organ.

Cardiac myocyte β3-adrenergic receptors prevent myocardial fibrosis by modulating oxidant stress-dependent paracrine signaling

Human and mouse cardiac beta3-adrenergic receptors (beta3AR) exert antipathetic effects to those of beta1-2AR stimulation. We examined their role in modulating myocardial remodelling, particularly fibrosis in response to haemodynamic stress. Mice with cardiac myocyte-specific expression of beta3AR (ADRB3-tg) or tamoxifen-inducible homozygous deletion (c-Adrb3-ko, with loxP-targeted Adrb3) were submitted to transaortic constriction. A superfusion assay was used for proteomic analysis of paracrine mediators between beta3AR-expressing cardiac myocytes and cardiac fibroblasts cultured separately. We show that cardiac beta3AR attenuate myocardial fibrosis in response to haemodynamic stress. Interstitial fibrosis and collagen content were reduced in ADRB3-tg, but augmented in c-Adrb3-ko. ADRB3 and collagen (COL1A1) expression were also inversely related in ventricular biopsies of patients with valve disease. Incubation of cardiac fibroblasts with media conditioned by hypertrophic myocytes induced fibroblast proliferation, myo-differentiation, and collagen production. These effects were abrogated upon ADRB3 expression in myocytes. Comparative shotgun proteomic analysis of the myocyte secretomes revealed a number of factors differentially regulated by beta3AR, among which connective tissue growth factor [CTGF (CCN2)] was prominently reduced. CTGF was similarly reduced in stressed hearts from ADRB3-tg, but increased in hearts from c-Adrb3-ko mice. CTGF expression was mediated by reactive oxygen species production which was reduced by ADRB3 expression in vitro and in vivo. This antioxidant and anti-fibrotic effect involved beta3AR coupling to the neuronal isoform of nitric oxide synthase (nNOS) in cardiac myocytes, as both were abrogated upon nNOS inhibition or Nos1 homozygous deletion. Cardiac beta3AR protect from fibrosis in response to haemodynamic stress by modulating nitric oxide and oxidant stress-dependent paracrine signaling to fibroblasts. Specific agonism at beta3AR may offer a new therapeutic modality to prevent cardiac fibrosis.

Chronic Hypobaric Hypoxia Induces Right Ventricular Hypertrophy and Apoptosis in Rats: Therapeutic Potential of Nanocurcumin in Improving Adaptation.

Nehra, Sarita, Varun Bhardwaj, Santosh Kar, and Deepika Saraswat. Chronic hypobaric hypoxia induces right ventricular hypertrophy and apoptosis in rats: therapeutic potential of nanocurcumin in improving adaptation. High Alt Med Biol. 17:342-352, 2016.-a sustained work load on the right heart on ascent to high altitudes promotes right ventricular hypertrophy (RVH), which eventually undergoes decompensation and promotes pathological damage. However, the exact set of events leading to damage remains unidentified. Curcumin is a natural antioxidant and antihypertrophic agent, but it has poor biostability. Nanotized curcumin (nanocurcumin) has emerged as a promising agent with improved biostability while retaining the therapeutic properties of curcumin. The present study aimed at analyzing the therapeutic properties of nanocurcumin in ameliorating cardiac damage due to chronic hypobaric hypoxia (HH)-induced RVH in comparison to curcumin. Sprague-Dawley rats exposed to HH (25,000 feet, effective oxygen fraction in air [FIO2] ∼0.08, temperature 28°C ± 1°C, relative humidity 55% ± 2% for 3, 7, 14, and 21 days) developed RVH with increased interstitial collagen content, Fulton's index, and cardiomyocyte cross-sectional area while upregulating atrial natriuretic peptide. Tissue damage due to apoptotic cell death was evident by cytochrome-c/caspase-3 activation and TUNEL assay. Concomitant modulation of cyclic guanosine monophosphate (cGMP)/cGK-1, calmodulin-dependent protein kinase II (CaMkinase II), and intracellular calcium levels with increased free radical-induced damage and lipid peroxidation further contributed to the right heart pathology. Nanocurcumin supplementation decreased HH-induced RVH and apoptosis while modulating cardiac cGMP/cGK-1 signaling, and maintaining CaMkinase II, intracellular calcium levels and redox status better than curcumin. Nanocurcumin-mediated antiapoptotic effects might have benefited residents and sojourners at high altitude in preventing hypoxic cardiac damage.

Simvastatin impairs the induction of pulmonary fibrosis caused by a western style diet: a preliminary study.

The role of an atherogenic diet in causing pulmonary fibrosis has received little attention and simvastatin has been shown to reduce pulmonary fibrosis in animal models. To determine if an atherogenic diet can induce pulmonary fibrosis and whether simvastatin treatment is beneficial by up-regulating heat shock protein 70 and 90. New Zealand white rabbits (n = 15) were divided: Group 1 (control); Group 2 (MC) received a normal rabbit diet with 1% methionine plus 0.5% cholesterol (atherogenic diet). Group 3 received the same diet as the MC group plus 5 mg/kg/day simvastatin orally (MCS). After 4 weeks, the lungs were collected and analysed. Picrosirus red staining of lung interstitial collagen content showed that the atherogenic diet increased fibrosis 2.9-fold (P < 0.05), bronchiole adventitial collagen was increased 2.3-fold (P < 0.05) and bronchiole epithelium was increased 34-fold (P < 0.05), and simvastatin treatment severely reduced this effect (P < 0.05). Western blot analysis showed that the atherogenic diet significantly reduced lung Hsp70 protein by 22% (P < 0.05) and Hsp90 protein by 18% (P < 0.05) and simvastatin treatment did not affect this result. However, aortic hyper-responsiveness to vasoconstrictors (angiotensin II and phenylephrine) were markedly reduced by simvastatin treatment. We report that an atherogenic diet stimulates pulmonary fibrosis and reduces lung Hsp70/Hsp90 protein concentration. Simvastatin impairs this by mechanisms unrelated to Hsp70/Hsp90, but possibly a reduction in angiotensin II receptor or alpha adrenergic receptor pathways. These results could have implications in idiopathic pulmonary fibrosis.

Prolyl-4-hydroxylase- 1 improves the stability of advanced plaques but accelerates the atherosclerotic lesion formation of early plaques

To investigate the changes in Prolyl 4-hydroxylase (P4Hα1) expression during the pathological course of atherosclerotic plaque development and to test the effect of P4Hα1 overexpression on vulnerability of early and advanced plaque in apolipoprotein E-deficient (ApoE−/−) mice. In vivo , the changes in P4Hα1 level were assessed during the pathological course of spontaneously induced atherosclerotic plaque in the aortic root of ApoE−/− mice at 10, 12, 14, and 16 weeks of age and fed a high-fat diet. A lentivirus-mediated P4Hα1 construct was transfected in carotid plaque induced in mice by perivascular collar placement for 2 and 10 weeks. After 2 weeks of lenti-P4Hα1 treatment, both early and advanced plaque exhibited stable characteristics, with increased collagen and smooth muscle cell content, reduced macrophage content, and no change in lipid content. Lenti-P4Hα1 treatment of early and advanced plaque substantially increased interstitial collagen content in the fibrous cap of plaque. It increased the carotid plaque size and the relative en face lesion area of the aorta in early but not advanced plaque, with reduced phosphatase and tensin homolog expression. Furthermore, the treatment lowered the expression of inflammatory cytokines and the production and activity of matrix metalloproteinase-9 and -2 in both early and advanced plaque. P4Hα1 overexpression had a differential effect at various stages of plaque pathological progression. P4Hα1 overexpression might be a valuable therapeutic modality in stabilizing advanced but not early plaque because of the adverse event of accelerated lesion formation.

The time window for therapy with peptide nanofibers combined with autologous bone marrow cells in pigs after acute myocardial infarction.

BackgroundWe previously showed that injection of peptide nanofibers (NF) combined with autologous bone marrow mononuclear cells (MNC) immediately after coronary artery ligation improves cardiac performance in pigs. To evaluate the clinical feasibility, this study was performed to determine the therapeutic time window for NF/MNC therapy in acute myocardial infarction (MI).Methods and ResultsA total of 45 adult minipigs were randomly grouped into 7 groups: sham or MI plus treatment with NS (normal saline), or NF or MNC alone at 1 day (1D) post-MI, or NF/MNC at 1, 4, or 7 days post-MI (N≥6). Cardiac function was assessed by echocardiography and ventricular catheterization. Compared with the NS control, pigs treated with NF/MNC at 1 day post-MI (NF/MC-1D) had the greatest improvement in left ventricle ejection fraction (LVEF; 55.1±1.6%; P<0.01 vs. NS) 2 months after MI. In contrast, pigs treated with either NF/MNC-4D or NF/MNC-7D showed 48.9±0.8% (P<0.05 vs. NS) and 43.5±2.3% (n.s. vs. NS) improvements, respectively. The +dP/dt and -dP/dt, infarct size and interstitial collagen content were also improved in the NF/MNC-1D and -4D groups but not in the -7D group. Mechanistically, MNC quality and the states of systemic inflammation and damaged heart tissue influence the therapeutic efficiency of NF/MNC therapy, as revealed by another independent study using 16 pigs.ConclusionsInjection of NF/MNC at 1 or 4 days, but not at 7 days post-MI, improves cardiac performance and prevents ventricular remodeling, confirming the importance of early intervention when using this therapy for acute MI.

Nestin is a Marker of Lung Remodeling Secondary to Myocardial Infarction and Type I Diabetes in the Rat

Upregulation of the intermediate filament protein nestin was identified in a subpopulation of fibroblasts during reactive and reparative fibrosis and directly contributed to the enhanced proliferative phenotype. The present study tested the hypothesis that nestin was expressed in lung fibroblasts and the pattern of expression represented a distinct marker of pulmonary remodeling secondary to myocardial infarction and type I diabetes. Nestin((+)) fibroblasts were detected in rat lungs and a subpopulation exhibited a myofibroblast phenotype delineated by the co-expression of smooth muscle α-actin. In the lungs of myocardial infarcted rats, interstitial collagen content and nestin mRNA/protein levels were significantly increased despite the absence of secondary pulmonary hypertension, whereas smooth muscle α-actin protein expression was unchanged. Exposure of rat pulmonary fibroblasts to pro-fibrotic stimuli angiotensin II and transforming growth factor-β significantly increased nestin protein levels. In the lungs of type I diabetic rats, the absence of a reactive fibrotic response was associated with a significant downregulation of nestin mRNA/protein expression. Nestin was reported a target of miR-125b, albeit miR-125b levels were unchanged in pulmonary fibroblasts treated with pro-fibrotic stimuli. Nestin((+)) cells lacking smooth muscle α-actin/collagen staining were also identified in rodent lungs and a transgenic approach revealed that expression of the intermediate filament protein was driven by intron 2 of the nestin gene. The disparate regulation of nestin characterized a distinct pattern of pulmonary remodeling secondary to myocardial infarction and type I diabetes and upregulation of the intermediate filament protein in lung fibroblasts may have facilitated in part the reactive fibrotic response.

ADAMTS13 modulates atherosclerotic plaque progression in mice via a VWF‐dependent mechanism

ADAMTS13 reduces the adhesiveness of hyperactive ultra-large von Willebrand factor (ULVWF) multimers by cleaving them into smaller, less active multimers. Recently, we and others have demonstrated that ADAMTS13 reduces atherosclerosis in hypercholesteremic apolipoprotein E (ApoE-/-) deficient mice. It is not known whether ADAMTS13 modulates atherosclerosis directly or indirectly by cleaving ULVWF multimers. We generated triple knockout Adamts13-/-/Vwf-/-/ApoE-/- mice to determine whether ADAMTS13 modulates atherosclerosis through its proteolytic effects on VWF or other potential mechanisms. Female mice were fed a high-fat Western diet beginning at 6weeks of age until they were sacrificed at 4months. We compared the extent of atherosclerosis in the serial cross-sections of the aortic sinus using the Verhoeff-Van Gieson stain. Macrophage and neutrophil infiltration were quantified by immunohistochemistry. Under plain polarized light interstitial collagen content in the serial cross-sections of the aortic sinus was quantified using picrosirius red stain. Deficiency of VWF in Adamts13-/-/ApoE-/- mice (Adamts13-/-/Vwf-/-/ApoE-/-) completely reversed exacerbated atherosclerosis (P<0.05 vs. Adamts13-/-/ApoE-/- mice). The lesion size, macrophage and neutrophil infiltration in the aortic sinus of Adamts13-/-/Vwf-/-/ApoE-/- mice were significantly decreased compared with Adamts13-/-/ApoE-/- mice (P<0.05), but similar to Vwf-/-/ApoE-/- mice. Additionally, interstitial collagen content in the aortic sinus of Adamts13-/-/Vwf-/-/ApoE-/- mice was significantly reduced compared with Adamts13-/-/ApoE-/- mice (P<0.05), but similar to Vwf-/-/ApoE-/- mice. Total cholesterol and triglyceride levels were similar among groups. ADAMTS13 modulates inflammatory plaque progression in hypercholesterolemic mice through a VWF-dependent mechanism. These findings provide further evidence on the pathophysiological role for the ADAMTS13/VWF axis in atherosclerosis.

The Effect of 2,3,4′,5-Tetrahydroxystilbene-2-O-β-D-Glucoside on Pressure Overload-Induced Cardiac Remodeling in Rats and Its Possible Mechanism

The aim of the present study was to investigate the effects of 2,3,4',5-tetrahydroxystilbene-2-O-beta-D-glucoside, an active component extracted from Polygonum multiflorum, on pressure overload-induced cardiac remodeling in rats. A rat model with cardiac remodeling was induced by abdominal aortic banding. 2,3,4',5-Tetrahydroxystilbene-2-O-beta-D-glucoside (30, 60, 120 mg/kg/day) was administered 3 days after abdominal aortic banding and continued for 30 days. The abdominal aortic banding-treated rats had significantly elevated blood pressure, left ventricular hypertrophy, and myocardial fibrosis. Left ventricular hypertrophy was characterized by an increase in the ratios of the heart and left ventricular weights to body weight, and increased myocyte cross-sectional areas, hypertrophic ventricular septum, and left ventricular posterior wall. The accumulation of myocardial interstitial perivascular collagen and elevated cardiac hydroxyproline content indicated myocardial fibrosis. The pathological changes above were attenuated by 2,3,4',5-tetrahydroxystilbene-2-O-beta-D-glucoside. Additionally, it markedly reduced collagen I and III expressions and regulated matrix metalloproteinase-2,9 and inhibitors of metalloproteinase expressions, as markers of myocardial fibrosis. Furthermore, we explored the underlying mechanisms for such effects of 2,3,4',5-tetrahydroxystilbene-2-O-beta-D-glucoside. The results showed that it significantly reduced myocardium angiotensin II, enhanced the activities of superoxide dismutase and glutathione peroxidase in serum and myocardial tissue, as well as inhibited protein expression of transforming growth factor-β1 and phosphorylation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase in the myocardial tissue. Our results suggest that 2,3,4',5-tetrahydroxystilbene-2-O-beta-D-glucoside could prevent cardiac remodeling induced by pressure overload in rats. The underlying mechanisms may be related to a decreasing angiotensin II level, an antioxidant effect of the tested compound, suppression of transforming growth factor-β1 expression, and inhibition of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase activation.