Interstitial Fibrosis Research Articles

Neuroimmune cross-talk in heart failure.

Heart failure (HF) is characterized by autonomic nervous system (ANS) imbalance and low-grade chronic inflammation. The bidirectional relationship between the ANS and immune system (IS) is named "neuroimmune cross-talk" (NICT), and is based on common signaling molecules, receptors, and pathways. NICT may be altered in HF, and neuroinflammation seems to be a main driver of HF progression. In HF, heightened sympathetic nerve activity triggers inflammatory cascades that lead to cardiomyocyte death and myocardial interstitial fibrosis. Concurrently, parasympathetic withdrawal may impair the cholinergic anti-inflammatory pathway, with a less effective immune response to infections or inflammatory events. Additionally, microglial activation and inflammatory molecules contribute to autonomic imbalance by acting on central nuclei and peripheral visceral feedbacks, which in turn promote adverse cardiac remodeling, HF decompensation, and potentially life-threatening arrhythmias. Therefore, neuroinflammation has been identified as a potential target for treatment. Pharmacological antagonism of the neurohormonal system remains the cornerstone of chronic HF therapy. While some drugs used in HF management may have additional benefits due to their anti-inflammatory properties, clinical trials targeting inflammation in patients with HF have so far produced inconclusive results. Nevertheless, considering the pathophysiological relevance of NICT, its modulation seems an appealing strategy to optimize HF management. Current research is therefore investigating novel pharmacological targets for anti-inflammatory drugs, and the immunomodulatory properties of denervation approaches and bioelectronic medicine devices targeting NICT and neuroinflammation in HF. A deeper understanding of the complex relationship between the ANS and IS, as outlined in this review, could therefore facilitate the design of future studies aimed at improving outcomes by targeting NICT in patients with HF.

ASK-1 activation exacerbates kidney dysfunction via increment of glomerular permeability and accelerates cellular aging in diabetic kidney disease model mice

Diabetic kidney disease (DKD) is a major disease characterized by early albuminuria and heightened risk of renal deterioration. Increased reactive oxygen species (ROS) production, especially in glomeruli, plays an important role in the progression of DKD. ROS also cause activation of Apoptosis signal-regulating kinase 1 (ASK-1), which is implicated in various organ injuries. However, the detailed mechanisms remain unclear. This study investigates ASK-1 activation in advanced DKD and its underlying mechanisms using GS442172, an ASK-1 inhibitor. In the DKD mouse model, activation of ASK-1 was observed. Although inhibition of ASK-1 activation improved hyperpermeability in glomerular endothelial cells. ASK-1 inhibition significantly reduced glomerular injury and albuminuria, while also attenuating tubular damage and interstitial fibrosis. RNA-seq analysis revealed an aging phenotype associated with ASK-1 activation in DKD. In vitro experiments demonstrated ASK-1 activation-induced cellular senescence in tubular cells via redox signaling. These results suggested that the critical role of ASK-1 activation in DKD pathogenesis, implicating glomerular injury, tubular damage, and cellular senescence. ASK-1 inhibitors are promising therapeutic strategies to mitigate the progression of DKD.

The Potential Role of Cigarette Smoke, Elastic Fibers, and Secondary Lung Injury in the Transition of Pulmonary Emphysema to Combined Pulmonary Fibrosis and Emphysema

Combined pulmonary fibrosis and emphysema (CPFE) is a distinct syndrome associated with heavy smoking. The fibrotic component of the disease is generally believed to be superimposed on previously existing pulmonary emphysema, but the mechanisms responsible for these changes remain poorly understood. To better understand the pathogenesis of CPFE, we performed a series of experiments that focused on the relationships between lung elastic fibers, cigarette smoke, and secondary lung injury. The results indicate that even brief smoke exposure predisposes the lung to additional forms of lung injury that may cause alveolar wall fibrosis. The proinflammatory activity of smoke-induced structural alterations in elastic fibers may contribute to this process by enhancing secondary lung inflammation, including acute exacerbations of chronic obstructive pulmonary disease. Furthermore, the levels of the unique elastin crosslinks, desmosine and isodesmosine, in blood, urine, and sputum may serve as biomarkers for the transition from pulmonary emphysema to interstitial fibrosis. While the long-term effects of these inflammatory reactions were not examined, the current studies provide insight into the potential relationships between elastic fiber injury, cigarette smoke, and secondary lung injury. Determining the mechanisms involved in combined pulmonary emphysema and fibrosis and developing a sensitive biomarker for this type of lung injury may permit timely therapeutic intervention that could mitigate the high risk of respiratory failure associated with this condition.

USP20 deletion promotes eccentric cardiac remodeling in response to pressure overload and increases mortality.

Left ventricular hypertrophy (LVH) caused by chronic pressure overload with subsequent pathological remodeling is a major cardiovascular risk factor for heart failure and mortality. The role of deubiquitinases in LVH has not been well characterized. To define whether the deubiquitinase ubiquitin-specific peptidase 20 (USP20) regulates LVH, we subjected USP20 knockout (KO) and cognate wild-type (WT) mice to chronic pressure overload by transverse aortic constriction (TAC) and measured changes in cardiac function by serial echocardiography followed by histological and biochemical evaluations. USP20-KO mice showed severe deterioration of systolic function within 4 wk of TAC compared with WT cohorts. Both USP20-KO TAC and WT-TAC cohorts presented cardiac hypertrophy following pressure overload. However, USP20-KO-TAC mice showed an increase in cardiomyocyte length and developed maladaptive eccentric hypertrophy, a phenotype generally observed with volume overload states and decompensated heart failure. In contrast, WT-TAC mice displayed an increase in cardiomyocyte width, producing concentric remodeling that is characteristic of pressure overload. In addition, cardiomyocyte apoptosis, interstitial fibrosis, and mouse mortality were augmented in USP20-KO-TAC compared with WT-TAC mice. Quantitative mass spectrometry of LV tissue revealed that the expression of sarcomeric myosin heavy chain 7 (MYH7), a fetal gene normally upregulated during cardiac remodeling, was significantly reduced in USP20-KO after TAC. Mechanistically, we identified increased degradative lysine-48 polyubiquitination of MYH7 in USP20-KO hearts, indicating that USP20-mediated deubiquitination likely prevents protein degradation of MYH7 during pressure overload. Our findings suggest that USP20-dependent signaling pathways regulate the layering pattern of sarcomeres to suppress maladaptive remodeling during chronic pressure overload and prevent cardiac failure.NEW & NOTEWORTHY We identify ubiquitin-specific peptidase 20 (USP20) as an important enzyme that is required for cardiac homeostasis and function, particularly during myocardial pressure overload. USP20 regulates protein stability of cardiac MYH7, an essential molecular motor protein expressed in sarcomeres; loss-of-function mutations of MYH7 are associated with human hypertrophic cardiomyopathy, cardiac failure, and sudden death. Enhancing USP20 activity could be a potential therapeutic approach to prevent the development of maladaptive state of eccentric hypertrophy and heart failure.

Polysaccharide isolated from Dioscorea septemloba improves hyperuricemia and alleviates renal fibrosis through gut-kidney axis in mice.

Hyperuricemia (HUA) is a common metabolic disorder that often accompanies kidney diseases such as tubule damage and renal interstitial fibrosis. The preventive and therapeutic effects of Dioscorea septemloba, an anti-HUA herb, polysaccharide of which was considered as the main active ingredient on HUA, need to be explored. The major polysaccharide component, BXP, was purified from Dioscorea septemloba, with an average molecular weight of 10.432 kDa. Structural analysis inferred that BXP backbone was composed of t-β-D-Glcp-(1 → 4)-α-D-Glcp-(1 → 4)-α-D-Glcp-(1 → 4, 3)-β-D-Glcp-(1→, along with the side chain of →1)-α-D-Glcp-(6, 4 → 3, 4)-β-D-Glcp-(1→. The HUA mouse model was further established to clarify the underlying effect of BXP on HUA alleviation. As results shown, BXP decreased serum uric acid by inhibiting XOD and regulating urate transporter expression (GLUT9, OAT3, OAT1, URAT1 and ABCG2) in HUA mice, as well as relieving kidney and liver damage. Moreover, results of microbiome and metabolomics indicated that BXP improved the abundance of gut bacteria and reversed the Lipids-related metabolism disorder caused by HUA. This study indicated that BXP had potential to alleviate HUA and kidney disease through the gut-kidney axis in mice.

Quantification of Replacement Fibrosis in Aortic Stenosis: A Narrative Review on the Utility of Cardiovascular Magnetic Resonance Imaging

Aortic stenosis (AS) is associated with the development of replacement myocardial fibrosis/scar. Given the dose-dependent relationship between scar and clinical outcomes after aortic valve replacement (AVR) surgery, scar quantity may serve as an important risk-stratification tool to aid decision-making on the optimal timing of AVR. Scar is non-invasively assessed and quantified by cardiovascular magnetic resonance (CMR) imaging. Several quantification techniques exist, and consensus on the optimal technique is lacking. These techniques range from a visual manual method to fully automated ones. This review describes the different scar quantification techniques used and highlights their strengths and shortfalls within the context of AS. The two most commonly used techniques in AS include the semi-automated signal threshold versus reference mean (STRM) and full-width half-maximum (FWHM) techniques. The accuracy and reproducibility of these techniques may be hindered in AS by the coexistence of diffuse interstitial fibrosis and the presence of relatively small, non-bright scars. The validation of these techniques against histology, which is the current gold standard for scar quantification in AS, is limited. Based on the best current evidence, the STRM method using a threshold of three standard deviations above the mean signal intensity of remote myocardium is recommended. The high reproducibility of the FWHM technique in non-AS cohorts has been shown and merits further evaluation within the context of AS. Future directions include the use of quantitative T1 mapping for the detection and quantification of scar, as well as the development of serum biomarkers that reflect the fibrotic status of the myocardium in AS.

Persistent Chronic Active T-Cell-Mediated Rejection After Kidney Transplantation Is Associated With Poor Allograft Survival.

Histopathological findings of chronic active T-cell-mediated rejection (CA-TCMR) have been reported to potentially improve with treatment. However, whether this improvement is associated with a better renal prognosis remains unclear. This study was performed to analyze the impact of the histological response to therapy on kidney allograft survival in patients with CA-TCMR. The data of patients diagnosed with CA-TCMR between January 2018 and May 2023 were retrospectively reviewed. A composite graft endpoint was defined as a two-fold increase in the serum creatinine level or the development of end-stage kidney disease. Thirty-seven patients with CA-TCMR underwent 46 follow-up biopsies. Eleven patients who were diagnosed with CA-TCMR at the last biopsy were classified as the persistent group, while the remaining 26 patients were classified as the transient group. Both before and after treatment, there were no significant changes in serum creatinine, estimated glomerular filtration rate, or proteinuria in either group. However, the transient group showed a significant reduction in interstitial fibrosis and tubular atrophy without a specific etiology (IFTA). This improvement was attributed to better histopathological Banff scores after treatment. Patients with persistent CA-TCMR had significantly worse graft survival than those with transient CA-TCMR (p = 0.002), even after adjusting for significant clinical factors (hazard ratio: 11.4; 95% CI: 1.1-120.0; p = 0.043). Our findings suggest that the persistence of histopathologic evidence of CA-TCMR after treatment is a significant risk factor for allograft loss compared with transient CA-TCMR.

Epigenetic BET inhibitor apabetalone counters inflammatory and fibrotic processes in activated cardiac fibroblasts providing insight into reduced hospitalizations for heart failure in BETonMACE trial

Abstract Background After myocardial infarction (MI), sustained crosstalk between monocytes and cardiac fibroblasts (CFs) promotes chronic inflammation and interstitial fibrosis that can contribute to heart failure (HF). Resident and myocardium-infiltrating immune cells produce proinflammatory cytokines IL-1β and TNFα that stimulate CFs to produce monocyte chemoattractants. Monocytes secrete TGF-β1 that transforms CFs into contractile myofibroblasts overproducing the extracellular matrix (ECM) responsible for cardiac fibrosis. Epigenetic BET inhibitors (BETi) reduce CF transdifferentiation to prevent cardiac fibrosis and HF in animal models. In patients with cardiovascular disease, type 2 diabetes and recent MI, administration of the BETi apabetalone (APA) resulted in less hospitalization due to HF vs. placebo (BETonMACE phase 3 trial; hazard ratio 0.59, p=0.03). Purpose To examine APA’s in vitro effects on inflammatory and profibrotic processes in CFs associated with cardiac fibrosis and HF. Methods Immortalized and primary human CFs were stimulated with cytokines (10ng/mL IL-1β, TNFα, TGF-β1) or with conditioned media from 10ng/mL IFNγ and 100ng/mL lipopolysaccharide-treated THP-1 macrophages ± 5μM APA on plastic or in 3D collagen gels. Gene expression was analyzed by PCR, protein levels by FACS or ELISA, collagen deposition with picrosirius red, THP-1 monocyte migration in Boyden chambers, and CF contraction with 3D collagen gels. Results IL-1β or TNFα stimulation of CFs upregulated monocyte chemoattractant 1 (CCL2) and vascular cell adhesion protein 1 (VCAM1) expression, promoting THP-1 cell migration and adhesion to CFs. APA treatment reduced cytokine-induced CCL2 (>20%) and VCAM1 (>70%) gene expression as well as THP-1 cell migration and adhesion to stimulated CFs (>60%). TGF-β1 treatment induced CF transdifferentiation into myofibroblasts as shown by increased expression of α smooth muscle actin (α-SMA) protein, and secretion of ECM proteins fibronectin and periostin. APA treatment reduced myofibroblast mRNA and protein expression of α-SMA (~30%), fibronectin (~30%) and periostin (97%). Myofibroblast-mediated collagen deposition was also reduced by APA by ~20%. α-SMA-dependent CF contraction can be visualized in attached 3D collagen gels. TGF-β1, IL-1β, TNFα or macrophage-conditioned media promoted CF-mediated gel contraction by 2.5, 1.4, 2.1, or 1.8-fold, respectively. APA treatment countered gel contraction by 30-60%, thereby reducing the myofibroblast-like behaviour in organotypic cultures. Conclusions In vitro, APA treatment reduced proinflammatory crosstalk between monocytes and CFs, resulting in less monocyte migration, monocyte - CF adhesion and CF contraction. APA also reduced signaling by TGF-β1, thus reducing profibrotic and contractile behaviour of CFs responsible for cardiac fibrosis. Since fibrosis contributes to HF, this data provides insight into the observed reduction in hospitalization due to HF in the BETonMACE trial.

Unraveling Marfan hearts: ultrastructural evidence for myocardial remodeling and cardiomyopathy

Abstract Background Despite improved life expectancy for patients with Marfan syndrome (MFS), significant morbidity and mortality persist. Although primarily attributed to aortic dissection and rupture, recent studies highlight the emerging role of arrhythmias and heart failure as additional contributors to morbidity and mortality in MFS. While severe valvular heart disease is an established cause of heart failure, there is a growing recognition of primary cardiomyopathy in patients with MFS. The mechanisms underlying cardiomyopathy in MFS remain poorly understood. Purpose This study aimed to reveal insights into the ultrastructural myocardial architecture in patients with MFS, thereby elucidating the role of cardiomyopathy in MFS. Methods This study enrolled 44 patients who underwent cardiac surgery at 2 centers (Marfan syndrome patients n=27 (MFS), ascending thoracic aortic aneurysm repair patients n=5 (non-MFS), patients post-orthotopic heart transplant n=12 (controls)). Myocardial biopsy samples were subjected to histopathological analysis using transmission electron microscopy and conventional histology. Results Conventional histological analysis revealed subtle abnormalities in the cardiomyocyte morphology of MFS patients, compared to non-MFS and controls. Overall, a slight increase in interstitial fibrosis and mild myocyte hypertrophy were observed in MFS patients. Ultrastructural examination of both MFS and non-MFS patients revealed significant structural remodeling and degenerative changes in the myocardium. These changes included: extensive myofibril lysis, compromised cell membrane integrity and nuclear chromatin condensation. Degenerative lesions were often accompanied by cytoplasmic debris, lysosomal residual bodies, autophagic vacuoles, and severe mitochondrial pathology. Dense accumulation of glycogen granules and lipofuscin was evident in areas of focal myofibril lysis. Notably, only MFS patients exhibited recruitment of granulocytes, including mast cells, eosinophils and neutrophils. A subset of MFS patients displayed advanced stages of myocardial degeneration. However, these changes did not correlate with clinical myocardial failure. Conclusions To the best of our knowledge, this study represents the first evidence of ultrastructural changes in the myocardium of patients with MFS. Our data indicate various degenerative changes in the myocardial architecture, including structural and metabolic myocardial remodeling, along with inflammatory cell infiltration. We believe that our findings of ultrastructural lesions reflect the complexity of myocardial disease in MFS and emphasize the importance of careful surveillance for cardiomyopathy in MFS patients. Further studies are warranted to confirm whether the observed granulocyte influx is specific to MFS and to clarify its role in MFS myocardial disease pathophysiology.Graphical abstract

Living with a chronic disease: tertiary prevention tools in asbestos-exposed patients

Abstract Background The main asbestos-related pathology is asbestosis, frequently associated with pleural plaques. The spirometric pattern is mainly restrictive, with reduction in all lung volumes (FEV1, FVC, TLC). Also a reduction in alveolus-capillary diffusion of carbon monoxide (DLCO) may be added, related to the fibrotic process. The aim of the study is to analyze the health surveillance data, correlating the spirometric finding and its variation over time with the clinical and radiological picture. Methods The study is conducted on 372 patients with asbestosis from occupational exposure diagnosed in the period 2017-2023, as part of the Tuscany Region’s program for former asbestos exposures (DGR 396/2016). The investigation is at the second level of surveillance, which includes chest CT and Respiratory Functionality Testing (RFT). Data are obtained from patients’ medical records and processed through a database. Results Out of 372 patients with asbestosis, 213 had pleural plaques (57.25%). Out of those, there is a progressive worsening of RFTs in a restrictive direction (TLC <80%, FEV1/FVC >70%) in 71 patients (33.3%), with a mean TLC loss of 8.7 ± 5.9 mL over subsequent follow-ups on a yearly basis. The FEV1/FVC ratio also declines, with a mean reduction of 5 ± 2.4%. Out of those 71 patients, 14 (19.7%) also show a mean reduction in DLCO of 6.7 ± 5 mL/min/mmHg. The negative trend in RFTs correlates with the worsening clinical and radiological picture attributable to the occurrence of plaques on a fibrotic framework. Conclusions The health surveillance protocol that we adopted has as objective the ascertainment of pulmonary pleura-parenchymal alterations correlated with occupational exposure, investigating their evolution over time through the analysis of spirometric data, and to optimize the management of the functional disability, with a view to the tertiary prevention of patients with asbestos-related disease. Key messages • The negative trend in asbestosis PFRs correlates with the worsening of the clinical and radiological picture attributable to the appearance of pleural plaques on an interstitial fibrosis. • Health surveillance is essential in order to optimize the management of functional disability, with a view to tertiary prevention of patients with asbestos-related disease.

The development of small-molecule ERBB4 agonists as a treatment for heart failure

Abstract The neuregulin-1 (NRG1)/ERBB4 signaling pathway has emerged as a cardioprotective pathway and is a promising target for the treatment of chronic heart failure. Activation of ERBB4 signaling is known to decrease cardiomyocyte cell death and hypertrophy, and fibroblast collagen synthesis. Recombinant NRG1 (rNRG1) is currently tested in phase III clinical trials for heart failure, but its need for intravenous administration is a disadvantage. In an attempt to circumvent this, we hypothesized that small-molecule-induced activation of ERBB4 is feasible and would recapitulate the effects of its natural ligand on myocytes and fibroblasts. To this end, we screened 10,240 compounds for their ability to induce homodimerization of ERBB4. We identified a series of 8 similar compounds (named EF-1 – EF-8) that concentration-dependently induced ERBB4 dimerization, with EF-1 being the most potent and effective compound (n = 4-5 independent repeats in each group; Emax = 27.9 ± 4.8% relative to NRG1, EC50 = 10.5 ± 4.5 x 10-6). EF-1 showed neither cytotoxicity nor increased cell proliferation of tumor cell lines. In vitro, EF-1 significantly decreased in a concentration-dependent manner hydrogen peroxide–induced cardiomyocyte cell death (n = 4 independent repeats in each group; P<0.0001 compared to siERBB4, Fig. 1A), angiotensin-II (AngII)-induced cardiomyocyte hypertrophy (n = 20 individual cardiomyocytes in each group; P<0.0001 compared to AngII/vehicle, Fig. 1B), and collagen expression in cultured human fibroblasts (n = 3 independent repeats in each group; P=0.03 compared to siERBB4, Fig. 1C). The observed effects in cultured cardiomyocytes and fibroblasts could be abrogated by siRNA targeting ERBB4, indicating that they were mediated by ERBB4. Moreover, when used in vivo, EF-1 (2 mg/kg/day) significantly decreased AngII-induced left ventricular Col1a1 and Col3a1 mRNA expression (n = 4-5 mice in each group; P=0.02 and P=0.004 compared to AngII/vehicle, respectively) and inhibited AngII-induced myocardial total and interstitial fibrosis in wild-type mice (n = 4-5 mice in each group, Fig. 2), but not in Erbb4-null mice. Moreover, EF-1 decreased acute cardiotoxicity (assessed by troponin release) in wild-type mice treated with doxorubicin (DOX; n = 8-9 mice in each group; P<0.0001 compared to DOX/vehicle), but not in Erbb4-null mice. In conclusion, we show that small-molecule-induced ERBB4 dimerization and activation is feasible, and recapitulates anti-fibrotic and cardiomyocyte protective effects in the heart in an ERBB4-dependent manner, both in vitro and in vivo. This could be the start for the further development of small-molecule ERBB4 agonists as a novel class of drugs to treat heart failure. Cardiomyocyte-protective effect in vitroAnti-fibrotic effect in vivo

Serial quantitative stress perfusion cardiac magnetic resonance in patients undergoing coronary artery bypass grafting; prediction of symptomatic benefit

Abstract Background Serial multiparametric cardiovascular magnetic resonance (CMR) in patients undergoing coronary artery bypass grafting (CABG) may provide mechanistic insight into dynamic and persistent abnormalities of the myocardium in patients with stable coronary artery disease (CAD). Objectives To assess for dynamic CMR markers of myocardial hypoperfusion and cardiac remodelling in patients undergoing CABG. Methods Patients with CAD awaiting elective CABG were recruited into an observational cohort study. Baseline evaluation included bloods, Seattle Angina Questionnaire-7, and adenosine stress perfusion CMR. Automated fully quantitative stress and rest myocardial blood flow was calculated, alongside assessment of the visual ischaemic burden. Native and post-contrast septal T1 indices were also measured. Repeat evaluation at 6-12 months post CABG was performed. Results Of 43 patients who underwent serial evaluation with CMR (mean age 62.1±9.3, median LVEF 68% [IQR: 62-73%]), there was improvement in the median visual ischaemic burden (18% [IQR: 11-21] vs 42% [IQR: 27-51], P<0.001), mean global stress myocardial blood flow (1.5±0.5 ml/min/g vs 1.3±0.5 ml/min/g, P=0.002) and median global myocardial perfusion reserve (2.4 [IQR: 1.7-2.8] vs 1.7 [IQR: 1.4-2.2], P=0.002) following CABG. Greater improvement in the SAQ-7 summary score was associated with a larger visual ischaemic burden at baseline (ρ=0.44, P=0.004) and a greater decrease in the visual ischaemic burden following CABG (ρ=-0.38, P=0.02). Quantitative MBF metrics did not associate with baseline or change in SAQ-7 summary score. Mean LV extracellular matrix volume decreased following CABG (16.6±3.6ml/m2 vs 18.2±4.6ml/m2, P=0.009). Conclusion Multiparametric CMR identifies dynamic changes in markers of myocardial perfusion and interstitial fibrosis in patients following CABG. Visual assessment of inducible myocardial hypoperfusion may identify patients who will derive the most symptomatic benefit from CABG. The results, however, suggest an important degree of residual inducible ischaemia and an unclear clinical role for CMR-derived MBF calculations.Serial CMR perfusion dataAssociation of SAQ with perfusion

Comparing the anti-remodeling effect of sacubitril/valsartan and angiotensin-converting enzyme inhibition on myocardial samples of advanced heart failure patients undergoing heart transplantation

Abstract Background In decreasing heart failure (HF) hospitalization and cardiovascular death in HF patients with reduced ejection fraction, Sacubitril/Valsartan (SAC/VAL) treatment was proved to be superior to angiotensin-converting enzyme inhibition (ACEi). According to recent experimental results and clinical data based on echocardiography and circulating biomarkers, this benefit might be at least partially attributed to a greater anti-remodeling effect. However, there is a lack of direct evidence from human myocardial samples for this assumption. Purpose Therefore, we aimed to compare the anti-remodeling effect of SAC/VAL treatment to ACEi on myocardial samples of HF patients undergoing heart transplantation (HTX). Methods We studied left ventricular (LV) samples of seventy advanced HF patients who underwent HTX and received either SAC/VAL (SAC/VAL group, N=32) or ACEi treatment (ACEi group, N=38) for at least three months prior to HTX. Furthermore, all patients received beta-blockers and mineralocorticoid receptor antagonists titrated to maximally tolerated or target dose. Our groups were matched for age, sex, ejection fraction and other clinical parameters. Patients with liver or kidney failure, mechanical circulatory support, systemic inflammatory/fibrotic diseases or positive inotrope treatment were excluded. Myocardial mRNA expression of fibrotic markers and B-type natriuretic peptide (BNP) were measured with qRT-PCR and normalized to glyceraldehyde 3-phosphate dehydrogenase expression. Interstitial fibrosis area was assessed histologically. Results Preoperative plasma N-terminal pro-BNP levels were similar in the two groups (SAC/VAL: 4363±752 pg/mL vs. ACEi: 4616±1020 pg/mL; p=0.911). In contrast, the mRNA level of BNP in the myocardium was lower in patients treated with SAC/VAL (SAC/VAL: 2.2±1.05 vs. ACEi: 5.2±1.74; p=0.006). TIMP metallopeptidase inhibitor 1 (SAC/VAL: 0.07±0.01 vs. ACEi: 0.13±0.02; p=0.011), TIMP metallopeptidase inhibitor 2 (SAC/VAL: 0.06±0.01 vs. ACEi: 0.11±0.01; p<0.001), collagen type I alpha 1 chain (SAC/VAL: 0.02±0.00 vs. ACEi: 0.08±0.02; p<0.001) and collagen type III alpha 1 chain (SAC/VAL: 0.12±0.02 vs. ACEi: 0.27±0.04; p<0.001) relative gene expressions were also decreased in the LV of SAC/VAL-treated patients. Histology analysis confirmed reduced interstitial myocardial fibrosis area in the SAC/VAL group as well (SAC/VAL: 7.2±0.4% vs. ACEi: 8.6±0.5%; p=0.031). Conclusions SAC/VAL treatment decreased the myocardial expression of BNP and fibrosis-related genes to a greater extent compared with ACEi in LV samples of patients undergoing HTX. Histological analysis also verified the differences in fibrosis. Thus, the anti-remodeling effect of SAC/VAL might be superior to ACEi even in advanced HF.

Relationships between 18-month kinetics and functional capacity and left ventricular remodeling and cardiac fibrosis in dilated cardiomyopathy

Abstract Introduction Myocardial fibrosis is a primary pathogenetic process in dilated cardiomyopathy (DCM) that is responsible for progressive cardiac remodeling and functional capacity impairment. We sought to verify whether there were differences between 18-month kinetics of functional capacity and left ventricular remodeling in DCM patients with different types and advancement of fibrosis who were up-titrated with guideline directed pharmacotherapy (GDMT). Methods Between May 2019 and September 2020, 99 DCM patients (88 male, mean age 45.2 ± 11.8 years, mean EF 29.7 ± 10%) underwent cardiac magnetic resonance with assessment of replacement fibrosis via late gadolinium enhancement (LGE) and interstitial fibrosis via extracellular volume (ECV). Each patient had serial (baseline, 6-, 12-, 18 month) functional assessment: NYHA class and 6- minute walking test (6-MWT) and follow-up echocardiography, including measurement of left ventricular end-diastolic volume (LVEDvol) and ejection fraction (EF). Patients were divided into LGE-negative and LGE-positive groups, whereas based on median ECV – they were divided into those with ECV below and above median values. Results Overall, LGE was identified in 44 (44%) of patients, whereas median ECV was 27.7%. NYHA class was significantly worse in patients with LGE (1.5±0.5 vs. 1.9±0.6) and with higher ECV (1.93±0.6 vs. 1.6±0.5) in comparison to those without LGE and lower ECV. However, NYHA class improved during observational period in all groups. There were no differences in 6-MWT distance in LGE positive and negative groups at all time points. Baseline 6-MWT distance in LGE positive group was 494.2±88.2 vs. 453.9±98.8 meters in LGE negative group. Baseline 6-MWT distance in upper median ECV group was (408.8±103.5 vs. 492.4±92.9 meter) was significantly lower ECV group (p=0.03). During 18 months 6-MWT distance increased only in LGE negative group but in both ECV groups (Figure 1 A-D). There were no differences in EF between patients with and without LGE and also with higher and lower ECV. Baseline EF in LGE positive group was 27.4±11.2% and 31.7±10.6% in LGE negative. Baseline indexed LVEDvol in LGE positive group was 107.7±40.7 vs. 91.8±37.8 ml/m2 (p=0.09) in LGE negative group. EF increased significantly in both groups, whereas indexed LVEDvol decreased only in LGE negative group (Figure 2 A-D). Conclusions Regardless of the presence of replacement and to some extent of interstitial fibrosis, functional and cardiac morphological improvement was observed during 18 months observation in DCM patients on GDMT. The greatest improvement occurred during the first 3 (occasionally 6) months which was associated with the greatest escalation of GDMT. Further dose escalation was not associated with a significant improvement in both functional and morphological parameters.

ERBB4 activation prevents microvascular dysfunction in a large animal model of hypertensive heart disease

Abstract Background Coronary microvascular dysfunction (CMD) participates in the pathophysiology of multiple cardiovascular diseases, but treatment options are limited. A new treatment option may include ERBB4 stimulation by neuregulin-1 (NRG1), which has anti-inflammatory, antifibrotic, and cardioprotective effects in models of heart failure. Objectives To assess the effect NRG1/ERBB4 stimulation on CMD in hypertensive heart disease. Methods Hypertensive heart disease was induced in 12 Aachener minipigs by implantation of deoxycorticosterone acetate (DOCA) pellets for 8 weeks and compared to 6 controls. The DOCA pigs were randomized to a weekly infusion of JK07, a NRG1 fusion protein with improved pharmacokinetic and pharmacodynamic properties, or vehicle. Microvascular resistance was measured using the bolus thermodilution method. Results DOCA significantly increased microvascular resistance compared to controls (from 14.5 to 19.9 mmHg.s, p = 0.028). This increase was abrogated by JK07 (11.3 mmHg.s, p = 0.018 vs DOCA). dP/dtmax increased by DOCA compared to controls (from 2415.5 to 4455.5 mmHg/s, p = 0.011), which was also abrogated by JK07 (3107.3 mmHg/s, p = 0.055 vs DOCA). Interstitial left ventricular fibrosis was significantly lower in JK07-treated pigs compared to DOCA only (2.1 vs. 5.4 %, p = 0.026), but without difference in perivascular fibrosis (p = 0.48). JK07 did not affect myocyte cross-sectional area, capillary density, pericyte coverage, or microvascular vessel thickness. Conclusions ERBB4 activation by JK07 is capable to prevent CMD in a DOCA hypertensive pig model. Functional rather than structural alterations may explain the protective effects of JK07 on microvascular resistance.Visual summaryJK07 decreases microvascular resistance

Tubulointerstitial injury in proteinuric chronic kidney diseases

Proteinuria is an independent risk factor for chronic kidney disease progression and cardiovascular diseases. Apart from its prognostic role, the load of proteins that pass across the disrupted glomerular capillary wall trigger multiple pathophysiologic processes. These include, among others, intratubular complement activation and excessive proximal tubular reabsorption of filtered proteins, especially albumin and albumin-bound free fatty acids, which can set off several pathways of cellular damage. The activation of these pathways can cause apoptosis of proximal tubular cells and paracrine effects that incite the development of interstitial inflammation and fibrosis, ultimately leading to irreversible kidney injury. In this review, we provide a comprehensive overview of the current understanding on the mechanisms underlying the tubular toxicity of ultrafiltered proteins in the setting of proteinuric chronic kidney diseases. The acquired knowledge is expected to be instrumental for the development of novel therapeutic classes of medications to be tested on top of standard of care with optimized renin-angiotensin-aldosterone blockade and sodium-glucose cotransporter-2 inhibition, in order to further improve the clinical outcomes of patients with proteinuric chronic kidney diseases.

Postoperative assessment of interstitial myocardial fibrosis in aortic valve pathologies: insights from a cardiac magnetic resonance study

Abstract Background Data on the characteristics of diffuse myocardial fibrosis (DMF) among patient undergoing valve surgery for aortic regurgitation (AR), high-gradient aortic stenosis (HG-AS), and low-flow low-gradient aortic stenosis with low ejection fraction (LFLG-AS) are limited. Purpose To assess the postoperative characteristics of DMF and to compare post- and pre-operative cardiac magnetic resonance imaging (CMR) data. Methods Patients diagnosed with AR (38), HG-AS (62), and LFLG-AS (39), who met criteria for surgical intervention, were prospectively included. Pseudo-severe AS was excluded. Post-operative CMR was performed 6 to 8 months after surgery, and a comparison between post- and pre-operative data was made (delta=Δ). Quantitative analysis included assessment of extracellular volume fraction (ECV), indexed ECV (iECV), and myocardial mass (MM). Results There were differences in baseline characteristics among patients with AR, HG-AS, and LFLG-AS concerning age (54±14 vs 63±8 vs 67±8 years, respectively; p<0.01), male gender (75% vs 50% vs 82%, respectively; p<0.01), NYHA functional class III and IV (37.5% vs 55.2% vs 30.8%, respectively; p=0.03), EuroSCORE II (1.16±0.5 vs 1.26±0.46 vs 3.39±2.65%, respectively; p<0.01), and STS (0.74±0.29 vs 1.11±0.5 vs 3.21±2.11%, respectively; p<0.01). Global ECV was similar between groups (29.2±5.6 vs 27.0±3.6 vs 29.4±5.9%, respectively; p=0.01), MM was different between groups (215±76 vs 161±51 vs 203±51g, respectively; p<0.01), and iECV was different between groups and significantly higher in AS patients (50.1±31.0 vs 21±8 vs 34.2±10.2ml/m², respectively; p<0.01). All patients underwent valve surgery and there were differences regarding extracorporeal circulation time (94±25 vs 97±20 vs 75±41min, respectively; p<0.01), and 30-day mortality (0% vs 3% vs 15%, respectively; p<0.01). Post-surgery data from patients with AR (32), HG-AS (62), and LFLG-AS (21) demonstrated a comparable decrease across all groups concerning preoperative MM (ΔMM: -39.3±41.5 vs -33.4±30.9 vs -23.4±34.2g, respectively; p=0.36) (Figure 1). In contrast, iECV decreased post-surgery only in AR, remaining stable in HG-AS and LFLG-AS (ΔiECV: -19.1±22.5 vs -1.4±7.4 vs 0.9±12ml/m², respectively; p<0.01). Thus, postoperative ECV maintained commensurate values in AR, while increasing in HG-AS and LFLG-AS (ΔECV: -0.1±4.5 vs 1.5±5.1 vs 2.9±4.9%, respectively; p=0.09). Δ left ventricular ejection fraction (LVEF) was similar across groups (ΔLVEF: -1.5±10.6 vs 1.1±11.7 vs 2.6±16%, respectively; p=0.44). Conclusion In the postoperative period, a decrease in MM was observed in all groups of aortic valve pathologies. However, only patients with AR showed a simultaneous and proportional reduction in DMF mass, explaining the patterns of ECV variation. Thus, the reduction of DMF in the postoperative period appears to vary according to the phenotypes presented, and patients with AR exhibit early reverse remodeling despite having high levels of DMF.Delta iECV, LV mass and ECV measures

A newly developed small-molecule ErbB4 agonist reduces reactive cardiac fibrosis and adverse ventricular remodelling after myocardial infarction in a sex-specific manner

Abstract Background The beneficial effects of the neuregulin/ErbB pathway on the diseased heart have been well-established. The anti-fibrotic and cardioprotective aspects of neuregulin are largely attributable to the activation of the ErbB4 receptor. We recently developed a small-molecule selective ErbB4 agonist, which reduced collagen production in cultured fibroblasts and prevented oxidative-stress induced cardiomyocyte death in vitro. Purpose In order to evaluate the in vivo effects of this small-molecule ErbB4 agonist (referred to as compound, Cpd), we assessed its effect on left ventricular remodelling, cardiac fibrosis and cardiac function in a murine model of myocardial infarction (MI). Since there is a potential interaction between ErbB4 and the oestrogen receptor, we also aimed to evaluate whether these effects were sex-dependent. We hypothesised that Cpd would attenuate left ventricular remodelling, reduce cardiac fibrosis and help retain cardiac function in a sex-specific manner. Methods Mice were divided by sex and then further divided into three experimental groups: a sham group, an MI group receiving Cpd (MI/Cpd), and a control MI group receiving vehicle only (MI/Veh). MI was induced by ligating the left descending coronary artery. Seven days after MI, mice started treatment with either Cpd (2mg/kg/d) or its vehicle through subcutaneous osmotic minipumps. Cardiac ultrasound was performed weekly until mice were sacrificed after four weeks of treatment. Hearts were excised and stained with Masson’s trichrome to assess reactive cardiac fibrosis in the myocardium of the left ventricle, located remote from the infarction scar. A WGA-isolectinB4-DAPI immunofluorescent stain was performed to assess cardiomyocyte cross-sectional area in order to evaluate cardiomyocyte hypertrophy. Results In females (n=25), Cpd significantly decreased left ventricular end-diastolic volume compared to vehicle (LVEDV, Fig.1A, 80±23 µl vs.108±29 µl, p=0.045) and induced a slightly increased left ventricular ejection fraction (LVEF, Fig.1B, 32±12% vs. 22±9%, p=0.086). In addition, Cpd significantly reduced reactive interstitial fibrosis myocardium (Fig.1C, fibrotic area, 1.4±1.0% vs. vehicle: 3.8±3.1%, p=0.0363). In males (n=30), Cpd had no significant effects on either of the parameters mentioned above, (Fig.1E-G). There was no significant treatment effect in either group on cardiomyocyte cross sectional area (Fig.1D, Fig.1H). Conclusion Treatment with the novel small-molecule ErbB4 agonist attenuates left ventricular dilation and reactive interstitial fibrosis after MI in female mice, but not in males. These data support the premise that small molecule-induced ErbB4 activation is a potential new therapy for heart failure, especially in female patients. The mechanisms underlying the sex-dependent effects warrant further exploration.Figure 1

Temporal dynamics of cardiac remodelling in pressure overload: the key role of visceral adipose tissue

Abstract Background Myocardial fibrosis is a prominent pathological feature of cardiac remodelling in pressure overload. The underlying mechanisms of cardiac remodelling are complex including mechanical, inflammatory and neurohormonal factors. We have recently uncovered a pathological crosstalk between visceral adipose tissue (VAT) and heart in biological aging characterized by profibrotic proteins in both the VAT secretome and the plasma contributing to myocardial interstitial fibrosis. We hypothesize that a model of pressure overload induced by transverse aortic constriction (TAC) is responsible for VAT remodelling and a VAT profibrotic secretome, which in turn worsens cardiac fibrosis and alters cardiac function. Purpose To explore the effect of pressure overload on VAT and identify a crosstalk between heart and VAT. Methods We divided wild-type male mice (5-month-old, n=10/group) into two groups: a visceral Lipectomy group and a Sham group two weeks before a moderate TAC (26G; i.e. gradient between left ventricle and aorta of 30mmHg) or a sham surgery. We sacrificed the mice at 1- and 8-week after TAC. We performed in vivo metabolic tests, echocardiography, and invasive hemodynamics, followed by ex vivo tissue analysis. We then analysed the time-course of the cardiac transcriptomic signature in TAC vs control and the impact of visceral lipectomy in both groups. Results Our results show that TAC induced not only cardiac remodelling affecting the structure and function of the heart, but also VAT fibrosis and inflammation together with activation of adrenergic receptors (Adrb3). VAT became a major source of profibrotic (TGFb and osteopontin) and proinflammatory (TNFa, IL6 and PAI1) factors as early as 1 week after TAC. Interestingly, lipectomy prevented cardiac remodelling and dysfunction. After 1 week of TAC, cardiac transcriptomic profile showed a similar upregulation of profibrotic and prohypertrophic genes in TAC groups in presence or absence of lipectomy. After 8 weeks of TAC, lipectomy prevented the upregulation of those genes and transcriptomic profile of TAC+lipectomy was similar to sham group (Figure). PCR analysis confirmed the lower expression of profibrotic (TGFb, Osteopontin), prohypertrophic (Nppa) and contractility genes (Adr1b) in TAC+Lipectomy vs TAC group after 8 weeks of TAC. Conclusion Our results suggest that VAT is detrimental to cardiac structure and function by releasing a profibrotic and a proinflammatory secretome that aggravates the cardiac remodelling induced by TAC. Conversely, lipectomy prevents the early development of TAC-induced cardiac fibrosis.

Subendocardial ischemic-like state in patients with severe aortic stenosis: insights from myocardial histopathology and ultrastructure

Abstract Background myocardial adaptation to severe aortic stenosis (AS) is a complex process that involves myocardial fibrosis (MF) beyond cardiomyocyte hypertrophy. Perfusion impairment is believed to be involved in myocardial remodeling in chronic pressure overload. Aim to describe morphological and ultrastructural myocardial changes at EMB, possibly reflecting subendocardial ischemia, in a group of patients with severe AS referred to surgical aortic valve replacement (AVR), with no previous history of ischemic cardiomyopathy. Methods one-hundred-fifty-eight patients (73 [68-77] years, 50% women) referred for surgical AVR because of severe symptomatic AS with pre-operative clinical and imaging study and no previous history of ischemic cardiomyopathy. Intra-operative septal EMB was obtained in 129 patients. Tissue sections were stained with Masson ́s Trichrome for MF quantification and periodic acid-Schiff (PAS) staining was performed to assess the presence of intracellular glycogen. Ultrastructure was analyzed through Transmission electron microscopy (TEM). Results MF totalized a median fraction of 11.90% [6.54-19.97%] of EMB, with highly prevalent perivascular involvement (95.3%). None of the samples had histological evidence of myocardial infarction. In 58 patients (45%) we found subendocardial groups of cardiomyocytes with cytoplasmatic enlargement, vacuolization and myofiber derangement, surrounded by extensive interstitial fibrosis. These cardiomyocytes were PAS positive, PAS-diastase resistant and Alcian Blue/PAS indicative of the presence of neutral intracellular glyco-saccharides. At TEM there were signs of cardiomyocyte degeneration with sarcomere disorganization and reduction, organelle rarefaction but no signs of intracellular specific accumulation. Conclusion almost half of the patients with severe AS referred for surgical AVR have histological and ultrastructural signs of subendocardial cardiomyocyte ischemic insult. It might be inferred that local perfusion imbalance contributes to myocardial remodeling and fibrosis in chronic pressure overload.