The Pathogenesis of Cardiac Fibrosis

HDAC6 Promotes Cardiac Fibrosis Progression through Suppressing RASSF1A Expression

DNA methylation in cardiac fibrosis: new advances and perspectives.

Heart disease causing cardiac cell death due to ischemia-reperfusion injury is a major cause of morbidity and mortality in the United States. Coronary heart disease and cardiomyopathies are the major cause for congestive heart failure, and thrombosis of the coronary arteries is the most common cause of myocardial infarction. Cardiac injury is followed by post-injury cardiac remodeling or fibrosis. Cardiac fibrosis is characterized by net accumulation of extracellular matrix proteins in the cardiac interstitium and results in both systolic and diastolic dysfunctions. It has been suggested by both experimental and clinical evidence that fibrotic changes in the heart are reversible. Hence, it is vital to understand the mechanism involved in the initiation, progression, and resolution of cardiac fibrosis to design anti-fibrotic treatment modalities. Animal models are of great importance for cardiovascular research studies. With the developing research field, the choice of selecting an animal model for the proposed research study is crucial for its outcome and translational purpose. Compared to large animal models for cardiac research, the mouse model is preferredby many investigators because of genetic manipulations and easier handling. This critical review is focused to provide insight to young researchers about the various mouse models, advantages and disadvantages, and their use in research pertaining to cardiac fibrosis and hypertrophy.

Angiotensin II plays an important role in the development of cardiac hypertrophy and fibrosis, but the underlying cellular and molecular mechanisms are not completely understood. Recent studies have shown that bone marrow-derived fibroblast precursors are involved in the pathogenesis of cardiac fibrosis. Since bone marrow-derived fibroblast precursors express chemokine receptor, CCR2, we tested the hypothesis that CCR2 mediates the recruitment of fibroblast precursors into the heart, causing angiotensin II-induced cardiac fibrosis. Wild-type and CCR2 knockout mice were infused with angiotensin II at 1,500 ng·kg(-1)·min(-1). Angiotensin II treatment resulted in elevated blood pressure and cardiac hypertrophy that were not significantly different between wild-type and CCR2 knockout mice. Angiotensin II treatment of wild-type mice caused prominent cardiac fibrosis and accumulation of bone marrow-derived fibroblast precursors expressing the hematopoietic markers, CD34 and CD45, and the mesenchymal marker, collagen I. However, angiotensin II-induced cardiac fibrosis and accumulation of bone marrow-derived fibroblast precursors in the heart were abrogated in CCR2 knockout mice. Furthermore, angiotensin II treatment of wild-type mice increased the levels of collagen I, fibronectin, and α-smooth muscle actin in the heart, whereas these changes were not observed in the heart of angiotensin II-treated CCR2 knockout mice. Functional studies revealed that the reduction of cardiac fibrosis led to an impairment of cardiac systolic function and left ventricular dilatation in angiotensin II-treated CCR2 knockout mice. Our data demonstrate that CCR2 plays a pivotal role in the pathogenesis of angiotensin II-induced cardiac fibrosis through regulation of bone marrow-derived fibroblast precursors.

Cardiac injury initiates repair mechanisms and results in cardiac remodeling and fibrosis, which appears to be a leading cause of cardiovascular diseases. Cardiac fibrosis is characterized by the accumulation of extracellular matrix proteins, mainly collagen in the cardiac interstitium. Many experimental studies have demonstrated that fibrotic injury in the heart is reversible; therefore, it is vital to understand different molecular mechanisms that are involved in the initiation, progression, and resolution of cardiac fibrosis to enable the development of antifibrotic agents. Of the many experimental models, one of the recent models that has gained renewed interest is isoproterenol (ISP)–induced cardiac fibrosis. ISP is a synthetic catecholamine, sympathomimetic, and nonselective β‐adrenergic receptor agonist. The overstimulated and sustained activation of β‐adrenergic receptors has been reported to induce biochemical and physiological alterations and ultimately result in cardiac remodeling. ISP has been used for decades to induce acute myocardial infarction. However, the use of low doses and chronic administration of ISP have been shown to induce cardiac fibrosis; this practice has increased in recent years. Intraperitoneal or subcutaneous ISP has been widely used in preclinical studies to induce cardiac remodeling manifested by fibrosis and hypertrophy. The induced oxidative stress with subsequent perturbations in cellular signaling cascades through triggering the release of free radicals is considered the initiating mechanism of myocardial fibrosis. ISP is consistently used to induce fibrosis in laboratory animals and in cardiomyocytes isolated from animals. In recent years, numerous phytochemicals and synthetic molecules have been evaluated in ISP‐induced cardiac fibrosis. The present review exclusively provides a comprehensive summary of the pathological biochemical, histological, and molecular mechanisms of ISP in inducing cardiac fibrosis and hypertrophy. It also summarizes the application of this experimental model in the therapeutic evaluation of natural as well as synthetic compounds to demonstrate their potential in mitigating myocardial fibrosis and hypertrophy.

Fendrr involves in the pathogenesis of cardiac fibrosis via regulating miR-106b/SMAD3 axis

Cardiac fibrosis: Cell biological mechanisms, molecular pathways and therapeutic opportunities

Granzyme B Deficiency Protects against Angiotensin II–Induced Cardiac Fibrosis

Cardiac fibrosis is characterized by net accumulation of extracellular matrix in the myocardium and is an integral component of most cardiac pathological conditions. Fibrotic remodeling of the ventricle has profound consequences on cardiac function. Increased deposition of interstitial collagen in the perimysial space is initially associated with a stiffer ventricle and diastolic dysfunction. At a later stage, accumulation of extracellular matrix proteins in the cardiac interstitium activates proteolytic pathways leading to the development of ventricular dilation and systolic failure. Disturbance of the matrix network in the fibrotic heart may cause systolic dysfunction through several distinct mechanisms. First, loss of fibrillar collagen may impair transduction of cardiomyocyte contraction into myocardial force development resulting in uncoordinated contraction of cardiomyocyte bundles. Second, disruption of key interactions between endomysial matrix proteins (eg, laminin and collagen) and their receptors in cardiomyocytes may promote cardiomyocyte death.1 Finally, fibrosis may result in sliding displacement (slippage) of cardiomyocytes leading to a decrease in the number of muscular layers in the ventricular wall and subsequent left ventricular dilation. Beyond its effects on cardiac function, fibrotic ventricular remodeling also promotes arrhythmogenesis through impaired anisotropic conduction and subsequent generation of reentry circuits. Extensive evidence suggests that hemodynamic overload activates the renin-angiotensin system triggering potent fibrogenic signals that stimulate cardiac fibroblasts and enhance collagen deposition in the myocardium. Angiotensin II, the central effector molecule of the renin-angiotensin system, stimulates fibroblast proliferation and promotes matrix protein synthesis through interactions involving the angiotensin II type 1 receptor. Angiotensin II–induced fibrosis appears to be mediated, at least in part, through activation of transforming growth factor (TGF)-β signaling pathways.2 Angiotensin II upregulates TGF-β …

Anti-oxidant and anti-inflammatory effects of ellagic and punicic acid in an in vitro model of cardiac fibrosis

Mast cells (MC) are innate immune cells present in virtually all body tissues with key roles in allergic disease and host defense. MCs recognize damage-associated molecular patterns (DAMPs) through expression of multiple receptors including Toll-like receptors and the IL-33 receptor ST2. MCs can be activated to degranulate and release pre-formed mediators, to synthesize and secrete cytokines and chemokines without degranulation, and/or to produce lipid mediators. MC numbers are generally increased at sites of fibrosis. They are potent, resident, effector cells producing mediators that regulate the fibrotic process. The nature of the secretory products produced by MCs depend on micro-environmental signals and can be both pro- and anti-fibrotic. MCs have been repeatedly implicated in the pathogenesis of cardiac fibrosis and in angiogenic responses in hypoxic tissues, but these findings are controversial. Several rodent studies have indicated a protective role for MCs. MC-deficient mice have been reported to have poorer outcomes after coronary artery ligation and increased cardiac function upon MC reconstitution. In contrast, MCs have also been implicated as key drivers of fibrosis. MC stabilization during a hypertensive rat model and an atrial fibrillation mouse model rescued associated fibrosis. Discrepancies in the literature could be related to problems with mouse models of MC deficiency. To further complicate the issue, mice generally have a much lower density of MCs in their cardiac tissue than humans, and as such comparing MC deficient and MC containing mouse models is not necessarily reflective of the role of MCs in human disease. In this review, we will evaluate the literature regarding the role of MCs in cardiac fibrosis with an emphasis on what is known about MC biology, in this context. MCs have been well-studied in allergic disease and multiple pharmacological tools are available to regulate their function. We will identify potential opportunities to manipulate human MC function and the impact of their mediators with a view to preventing or reducing harmful fibrosis. Important therapeutic opportunities could arise from increased understanding of the impact of such potent, resident immune cells, with the ability to profoundly alter long term fibrotic processes.

Diabetic nephropathy (DN) is a major microvascular complication of diabetes, representing the most common cause of end stage renal failure. DN is considered to contribute to the increased morbidity and mortality observed in patients with diabetes. In diabetes a range of hemodynamic and metabolic factors interact leading to the development and progression of DN. The early stages of DN are clinically characterised by excessive urinary excretion of albumin with a subsequent decline in glomerular filtration rate (GFR) in more advanced stages of DN leading to end stage renal failure. It has been suggested that microalbuminuria in diabetic patients may be associated with intra-glomerular hyperfiltration early in DN and possibly also reduced tubular reabsorption, although the exact cause remains to be determined. The characteristic histopathological changes in DN include renal hypertrophy, the subsequent development of thickened glomerular and tubular basement membranes and the progressive accumulation of extracellular matrix (ECM) proteins in the glomerular mesangium and tubulointerstitium resulting in glomerulosclerosis and tubulointerstitial fibrosis ultimately leading to end stage renal insufficiency. The underlying mechanisms responsible for diabetic nephropathy remain poorly understood. Therefore, more effective and mechanism-based therapies are needed. In recent years, it has been postulated that diabetes mellitus is associated with increased renal oxidative stress, more specifically, increased levels of reactive oxygen species (ROS), resulting in renal damage. Accordingly, oxidative stress is increasingly considered to be a major contributor to the development and progression of DN. Various renal sources of ROS have been suggested to be relevant to the diabetic kidney. However, NADPH oxidases (Nox) are the only known dedicated source of the ROS forming enzyme family. Nox are suggested to play a pivotal role in the development and progression of renal injury in animal models of type 1 and type 2 diabetic nephropathy and hence, they represent a potentially important novel target. In the rodent kidney three isoforms of the catalytic subunit (Nox1, Nox2 and Nox4) of NADPH oxidase are expressed. However, the relative importance of the Nox1, 2 and 4 isoforms in the development and progression of diabetic nephropathy remains unclear. With respect to Nox2, our own studies in streptozotocin-induced diabetic Nox2 knockout (KO) mice have shown increased susceptibility to infections and 100% mortality at week 20 of diabetes. Thus, it was not considered that Nox2 blockade should be a priority in the studies presented in this thesis which address strategies to reduce diabetic nephropathy. Nox4, originally termed Renox, is highly expressed in renal tissues. The role of Nox4 in DN remains controversial. Nox4 downregulation by systemic administration of antisense oligonucleotides, albeit for a short period of only 2 weeks, reduced renal and glomerular hypertrophy and attenuated the increased expression of fibronectin in renal cortex and glomeruli in streptozotocin-induced diabetic rats. However, the Nox4 antisense oligonucleotide may not be absolutely specific for Nox4. On the other hand, a study by Babelova et al has found no upregulation of Nox4 in diabetic C57/bl6 mice compared to its non-diabetic counterpart, and Nox4 deficiency did not attenuate nephropathy. In the non-diabetic context using unilateral ureteric obstruction, an experimental model of renal injury leading to interstitial fibrosis, Nox4 deficiency in C57/bl6 mice was associated with increased renal fibrosis, tubular atrophy and increased ROS formation, consistent with a potential protective role for Nox4. With respect to Nox1, this isoform appears to play a major role in diabetic macrovascular disease but not much is known about the role of Nox1 in diabetic nephropathy. Thus, it remains to be determined which Nox isoform plays the most important role in diabetic kidney disease. We hypothesized that genetic deletion of the NADPH oxidase isoforms, Nox1 or Nox4 or specific pharmacological inhibition of these isoforms would confer various degrees of reno-protection in an animal model of diabetes associated renal disease. Furthermore, it is hypothesized that Nox4 deletion specifically in podocytes, a cell type in the kidney implicated with the pathogenesis of glomerular albumin leakage would attenuate albuminuria and renal injury in DN in vivo. Furthermore, it was postulated that in vitro Nox4 silencing in human podocytes would attenuate pro-inflammatory and pro-fibrotic pathways characteristic of the diabetic milieu. In this light, the overall objective of this thesis was to examine the role of the Nox1 and Nox4 isoforms of NADPH oxidase in a model of diabetic nephropathy in vivo as well as to investigate the mechanisms providing renoprotection using in vitro approaches. The first chapter of this thesis represents the introduction and literature review (Chapter 1), which collectively cover the following central themes addressed in this study. ‘Part A’ of the literature review addresses the general introduction of diabetes, the pathophysiology of diabetic nephropathy, oxidative stress, ROS, NADPH oxidase including its various isoforms Nox1, Nox4, Nox2 and Nox5 and their respective role and regulation in diabetic nephropathy. ‘Part B’ of the literature review is a published review paper 1 entitled “NAD(P)H oxidase isoforms as therapeutic targets for diabetic complications” which addresses the pathophysiological role of the different Nox isoforms in vascular complications of diabetes including diabetic nephropathy. This manuscript also summarises the most recent and relevant Nox specific therapeutic agents including GKT137831 (Genkyotex, Geneva, Switzerland) for the prevention and treatment of diabetic nephropathy. GKT137831 is a novel, first in class Nox1/Nox4 specific inhibitor which has been used for pharmacological intervention in my research project. Furthermore, ‘Part C’ of the literature review is a published review paper 2 entitled “Identifying and interpreting novel targets that address more than one diabetic complication: a strategy for optimal end organ protection in diabetes”. This review paper provides an overview of diabetic complications including diabetic nephropathy together with various therapeutic agents currently available or in development including Nox specific inhibitors for the prevention and treatment of DN in different experimental settings. Chapter 2 describes the expanded methodology used for laboratory experimental investigations. The succeeding chapters 3 and 4 describe the outcome of my laboratory experimental investigations. Chapter 3 is a published original research paper titled “Genetic Targeting or Pharmacologic Inhibition of NADPH Oxidase Nox4 Provides Renoprotection in Long-Term Diabetic Nephropathy”. This manuscript addresses the main questions of my PhD project. I have used global genetically modified animal models as well as pharmacological inhibition approaches to explore the role of Nox1 and Nox4 NADPH oxidase isoforms in an experimental model of diabetic nephropathy in vivo and in conditionally immortalized human podocyes in vitro. I have been able to investigate for the first time the long-term effects of Nox1 and Nox4 deletion in the development and progression of diabetic nephropathy by directly comparing renal injury in streptozotocin (STZ)-induced diabetic Nox1¯/? ApoE-/- and Nox4-/-ApoE-/- double knockout mice and their respective wild type (WT) control mice. In addition, the genetic deletion studies were complemented by a pharmacological intervention study using the currently most specific Nox inhibitor, GKT137831. Key findings in the in vivo studies were also addressed in vitro by using conditionally immortalized human renal epithelial cells also known as podocytes which are a distinct cell population implicated in the regulation of albuminuria. The laboratory findings which I have described are novel and demonstrate that Nox4 is the main source of renal ROS in a mouse model of diabetic nephropathy induced by streptozotocin administration in ApoE-/- mice. Deletion of Nox4 but not Nox1 resulted in renal protection, specifically reduced glomerular injury as evidenced by attenuated albuminuria, preserved renal structure, reduced glomerular accumulation of extracellular matrix proteins, attenuated glomerular macrophage infiltration and reduced renal expression of inflammatory markers such as monocyte chemoattractant protein-1 and the transcription factor NF-kB. Importantly, administration of the specific Nox1/4 inhibitor, GKT137831 to diabetic ApoE-/- mice, replicated these renoprotective effects of Nox4 deletion. In human podocytes, silencing of the Nox4 gene resulted in reduced production of ROS and downregulation of proinflammatory (MCP-1 and NF-kB) and profibrotic proteins (collagen IV and fibronectin) which are implicated in diabetic nephropathy. In addition, chapter 4 represents an unpublished manuscript entitled “Podocyte specific Nox4 deletion attenuates albuminuria in diabetic nephropathy”. I have successfully demonstrated the detrimental role of Nox4 in the development and progression of diabetic nephropathy using a global Nox4KO mouse model. Albuminuria is a key feature of diabetic nephropathy and it is considered that injury to podocytes is associated with the development of albuminuria. The podocyte, a glomerular epithelial cell, is a critical cell type in the slit pore diaphragm and protects from protein leakage. Podocyte injury is characterised by foot process effacement, increased VEGF and decreased nephrin expression, as well as loss of podocytes in the urine ultimately resulting in the development of albuminuria. In this light, I have explored the role of Nox4 in podocytes in a model of diabetic nephropathy. esis.

Myocardial fibrosis is closely associated with severe cardiovascular diseases, characterized by increased mortality rates worldwide. The development of myocardial fibrosis is based on the differentiation of fibroblasts into myofibroblasts, which synthesize components of the extracellular matrix in excess. A key regulator of fibroblast differentiation into myofibroblasts is transforming growth factor-beta. In recent years, close attention in the pathogenesis of fibrosis has been given to other growth factors – neurotrophins. It was recently discovered that fibroblasts express brain-derived neurotrophic factor (BDNF), and its receptors are involved in the pathogenesis of fibrosis of various organs and tissues. The role of BDNF and its receptors in the pathogenesis of myocardial fibrosis is just beginning to be studied. This review summarizes the information available in the literature (2019-2023) on the pathophysiological and pathogenetic mechanisms of the relationship between BDNF and cardiac fibrosis. Data presented in the literature showed that the mechanisms of action of BDNF in the cardiovascular system and the pathogenesis of cardiac fibrosis have common points of intersection, which makes this neurotrophin a promising therapeutic target for cardiac fibrosis. Further investigation of these aspects will allow the use of various external effects of BDNF to develop technology for the prevention of cardiac fibrosis.

PLK2 is a novel regulator of osteopontin-driven fibrosis and diastolic dysfunction in permanent atrial fibrillation

Increased Tenascin-C expression contributes to cardiac dysfunction and fibrosis in Duchenne muscular dystrophy