Mohamad Azhar

Origin of Cardiac Fibroblasts and the Role of Periostin

Cardiac fibroblasts are the most populous nonmyocyte cell type within the mature heart and are required for extracellular matrix synthesis and deposition, generation of the cardiac skeleton, and to electrically insulate the atria from the ventricles. Significantly, cardiac fibroblasts have also been shown to play an important role in cardiomyocyte growth and expansion of the ventricular chambers during heart development. Although there are currently no cardiac fibroblast-restricted molecular markers, it is generally envisaged that the majority of the cardiac fibroblasts are derived from the proepicardium via epithelial-to-mesenchymal transformation. However, still relatively little is known about when and where the cardiac fibroblasts cells are generated, the lineage of each cell, and how cardiac fibroblasts move to reside in their final position throughout all four cardiac chambers. In this review, we summarize the present understanding regarding the function of Periostin, a useful marker of the noncardiomyocyte lineages, and its role during cardiac morphogenesis. Characterization of the cardiac fibroblast lineage and identification of the signals that maintain, expand and regulate their differentiation will be required to improve our understanding of cardiac function in both normal and pathophysiological states.

Transforming growth factor beta in cardiovascular development and function

Transforming growth factor betas (TGFbetas) are pleiotropic cytokines involved in many biological processes. Genetic engineering and tissue explanation studies have revealed specific non-overlapping roles for TGFbeta ligands and their signaling molecules in development and in normal function of the cardiovascular system in the adult. In the embryo, TGFbetas appear to be involved in epithelial-mesenchymal transformations (EMT) during endocardial cushion formation, and in epicardial epithelial-mesenchymal transformations essential for coronary vasculature, ventricular myocardial development and compaction. In the adult, TGFbetas are involved in cardiac hypertrophy, vascular remodeling and regulation of the renal renin-angiotensin system. The evidence for TGFbeta activities during cardiovascular development and physiologic function will be given and areas which need further investigation will be discussed.

Dual Roles of Immunoregulatory Cytokine TGF-β in the Pathogenesis of Autoimmunity-Mediated Organ Damage

Ample evidence suggests a role of TGF-β in preventing autoimmunity. Multiorgan inflammatory disease, spontaneous activation of self-reactive T cells, and autoantibody production are hallmarks of autoimmune diseases, such as lupus. These features are reminiscent of the immunopathology manifest in TGF-β1-deficient mice. In this study, we show that lupus-prone (New Zealand Black and White)F1 mice have reduced expression of TGF-β1 in lymphoid tissues, and TGF-β1 or TGF-β1-producing T cells suppress autoantibody production. In contrast, the expression of TGF-β1 protein and mRNA and TGF-β signaling proteins (TGF-β receptor type II and phosphorylated SMAD3) increases in the target organs, i.e., kidneys, of these mice as they age and develop progressive organ damage. In fact, the levels of TGF-β1 in kidney tissue and urine correlate with the extent of chronic lesions that represent local tissue fibrosis. In vivo TGF-β blockade by treatment of these mice with an anti-TGF-β Ab selectively inhibits chronic fibrotic lesions without affecting autoantibody production and the inflammatory component of tissue injury. Thus, TGF-β plays a dual, seemingly paradoxical, role in the development of organ damage in multiorgan autoimmune diseases. According to our working model, reduced TGF-β in immune cells predisposes to immune dysregulation and autoantibody production, which causes tissue inflammation that triggers the production of anti-inflammatory cytokines such as TGF-β in target organs to counter inflammation. Enhanced TGF-β in target organs, in turn, can lead to dysregulated tissue repair, progressive fibrogenesis, and eventual end-organ damage.

Ligand-specific function of transforming growth factor beta in epithelial-mesenchymal transition in heart development.

The ligand specificity of transforming growth factor beta (TGFβ) in vivo in mouse cardiac cushion epithelial‐to‐mesenchymal transition (EMT) is poorly understood. To elucidate the function of TGFβ in cushion EMT, we analyzed Tgfb1−/−, Tgfb2−/−, and Tgfb3−/− mice between embryonic day (E) 9.5 and E14.5 using both in vitro and in vivo approaches. Atrioventricular (AV) canal collagen gel assays at E9.5 indicated normal EMT in both Tgfb1−/− and Tgfb3−/− mice. However, analysis of Tgfb2−/− AV explants at E9.5 and E10.5 indicated that EMT, but not cushion cell proliferation, was initially delayed but later remained persistent. This was concordant with the observation that Tgfb2−/− embryos, and not Tgfb1−/− or Tgfb3−/− embryos, develop enlarged cushions at E14.5 with elevated levels of well‐validated indicators of EMT. Collectively, these data indicate that TGFβ2, and not TGFβ1 or TGFβ3, mediates cardiac cushion EMT by promoting both the initiation and cessation of EMT. Developmental Dynamics 238:431–442, 2009.

Biological functions of the low and high molecular weight protein isoforms of fibroblast growth factor‐2 in cardiovascular development and disease

Fibroblast growth factor 2 (FGF2) consists of multiple protein isoforms (low molecular weight, LMW, and high molecular weight, HMW) produced by alternative translation from the Fgf2 gene. These protein isoforms are localized to different cellular compartments, indicating unique biological activity. FGF2 isoforms in the heart have distinct roles in many pathological circumstances in the heart including cardiac hypertrophy, ischemia–reperfusion injury, and atherosclerosis. These studies suggest distinct biological activities of FGF2 LMW and HMW isoforms both in vitro and in vivo. Yet, due to the limitations that only the recombinant FGF2 LMW isoform is readily available and that the FGF2 antibody is nonspecific with regards to its isoforms, much remains to be determined regarding the role(s) of the FGF2 LMW and HMW isoforms in cellular behavior and in cardiovascular development and pathophysiology. This review summarizes the activities of LMW and HMW isoforms of FGF2 in cardiovascular development and disease. Developmental Dynamics 238:249–264, 2009.

The neural crest is contiguous with the cardiac conduction system in the mouse embryo: a role in induction?

In this study we present data on the spatial relationship between neural crest-derived cells (NCC) and the specialized cardiac conduction system (CCS) in the developing murine heart. Using Wnt1-Cre/R26R conditional reporter mice that express β-galactosidase from ROSA26 upon Cre-mediated recombination, two populations of NCC are seen: one migrates through the arterial pole and contributes to the bundle branches, whereas the second population enters by way of the venous pole and provides cells to the sinoatrial and atrioventricular node areas. The CCS/lacZ construct is found in the myocardium of the early embryonic heart and afterward only persists in the definitive CCS and is acknowledged as a reporter for the developing conduction system. The contiguous expression of both reporters is suggestive for a potential role of cardiac NCC in the induction of the final differentiation of the CCS.

Transforming growth factor beta signaling in adult cardiovascular diseases and repair

The majority of children with congenital heart disease now live into adulthood due to the remarkable surgical and medical advances that have taken place over the past half century. Because of this, adults now represent the largest age group with adult cardiovascular diseases. It includes patients with heart diseases that were not detected or not treated during childhood, those whose defects were surgically corrected but now need revision due to maladaptive responses to the procedure, those with exercise problems and those with age-related degenerative diseases. Because adult cardiovascular diseases in this population are relatively new, they are not well understood. It is therefore necessary to understand the molecular and physiological pathways involved if we are to improve treatments. Since there is a developmental basis to adult cardiovascular disease, transforming growth factor beta (TGFβ) signaling pathways that are essential for proper cardiovascular development may also play critical roles in the homeostatic, repair and stress response processes involved in adult cardiovascular diseases. Consequently, we have chosen to summarize the current information on a subset of TGFβ ligand and receptor genes and related effector genes that, when dysregulated, are known to lead to cardiovascular diseases and adult cardiovascular deficiencies and/or pathologies. A better understanding of the TGFβ signaling network in cardiovascular disease and repair will impact genetic and physiologic investigations of cardiovascular diseases in elderly patients and lead to an improvement in clinical interventions.

Transforming growth factor Beta2 is required for valve remodeling during heart development

Although the function of transforming growth factor beta2 (TGFβ2) in epithelial mesenchymal transition (EMT) is well studied, its role in valve remodeling remains to be fully explored. Here, we used histological, morphometric, immunohistochemical and molecular approaches and showed that significant dysregulation of major extracellular matrix (ECM) components contributed to valve remodeling defects in Tgfb2−/− embryos. The data indicated that cushion mesenchymal cell differentiation was impaired in Tgfb2−/− embryos. Hyaluronan and cartilage link protein‐1 (CRTL1) were increased in hyperplastic valves of Tgfb2−/− embryos, indicating increased expansion and diversification of cushion mesenchyme into the cartilage cell lineage during heart development. Finally, Western blot and immunohistochemistry analyses indicate that the activation of SMAD2/3 was decreased in Tgfb2−/− embryos during valve remodeling. Collectively, the data indicate that TGFβ2 promotes valve remodeling and differentiation by inducing matrix organization and suppressing cushion mesenchyme differentiation into cartilage cell lineage during heart development. Developmental Dynamics 240:2127–2141, 2011.

The Inter-Relationship of Periostin, TGFβ, and BMP in Heart Valve Development and Valvular Heart Diseases

Recent studies have suggested an important role for periostin and transforming growth factor beta (TGFβ) and bone morphogenetic protein (BMP) ligands in heart valve formation and valvular heart diseases. The function of these molecules in cardiovascular development has previously been individually reviewed, but their association has not been thoroughly examined. Here, we summarize the current understanding of the association between periostin and TGFβ and BMP ligands, and discuss the implications of this association in the context of the role of these molecules in heart valve development and valvular homeostasis. Information about hierarchal connections between periostin and TGFβ and BMP ligands in valvulogenesis will increase our understanding of the pathogenesis, progression, and medical treatment of human valve diseases.

Adaptation of a planar microbiaxial optomechanical device for the tubular biaxial microstructural and macroscopic characterization of small vascular tissues.

Murine models of disease are a powerful tool for researchers to gain insight into disease formation, progression, and therapies. The biomechanical indicators of diseased tissue provide a unique insight into some of these murine models, since the biomechanical properties in scenarios such as aneurysm and Marfan syndrome can dictate tissue failure and mortality. Understanding the properties of the tissue on the macroscopic scale has been shown to be important, as one can then understand the tissues ability to withstand the high stresses seen in the cardiac pulsatile cycle. Alterations in the biomechanical response can foreshadow prospective mechanical failure of the tissue. These alterations are often seen on the microstructural level, and obtaining detailed information on such changes can offer a better understanding of the phenomena seen on the macroscopic level. Unfortunately, mouse models present problems due to the size and delicate features in the mechanical testing of such tissues. In addition, some smaller arteries in large-animal studies (e.g., coronary and cerebral arteries) can present the same issues, and are sometimes unsuitable for planar biaxial testing. The purpose of this paper is to present a robust method for the investigation of the mechanical properties of small arteries and the classification of the microstructural orientation and degree of fiber alignment. This occurs through the cost-efficient modification of a planar biaxial tester that works in conjunction with a two-photon nonlinear microscope. This system provides a means to further investigate how microstructure and mechanical properties are modified in diseased transgenic animals where the tissue is in small tube form. Several other hard-to-test tubular specimens such as cerebral aneurysm arteries and atherosclerotic coronary arteries can also be tested using the described modular device.