Mohammad T. Elnakish

Stem cell transplantation as a therapy for cardiac fibrosis

Cardiac fibrosis is a fundamental constituent of most cardiac pathologies and represents the upshot of nearly all types of cardiac injury. Generally, fibrosis is a scarring process, characterized by accumulation of fibroblasts and deposition of increasing amounts of extracellular matrix (ECM) proteins in the myocardium. Therapeutic approaches that control fibroblast activity and evade maladaptive processes could represent a potential strategy to attenuate progression towards heart failure. Currently, cell therapy is actively perceived as an alternative to traditional pharmacological management of myocardial infarction (MI). The majority of the studies applying stem cell therapy following MI have demonstrated a decline in fibrosis. However, it was not clearly recognized whether the decline in cardiac fibrosis was due to replacement of dead cardiomyocytes or because of the direct effects of paracrine factors released from the transplanted stem cells on the ECM. Therefore, the main focus of this review is to discuss the impact of different types of stem cells on cardiac fibrosis and associated cardiac remodelling in a variety of experimental models of heart failure, particularly MI.

Mesenchymal Stem Cells for Cardiac Regeneration: Translation to Bedside Reality

Cardiovascular disease (CVD) is the leading cause of death worldwide. According to the World Health Organization (WHO), an estimate of 17.3 million people died from CVDs in 2008 and by 2030, the number of deaths is estimated to reach almost 23.6 million. Despite the development of a variety of treatment options, heart failure management has failed to inhibit myocardial scar formation and replace the lost cardiomyocyte mass with new functional contractile cells. This shortage is complicated by the limited ability of the heart for self-regeneration. Accordingly, novel management approaches have been introduced into the field of cardiovascular research, leading to the evolution of gene- and cell-based therapies. Stem cell-based therapy (aka, cardiomyoplasty) is a rapidly growing alternative for regenerating the damaged myocardium and attenuating ischemic heart disease. However, the optimal cell type to achieve this goal has not been established yet, even after a decade of cardiovascular stem cell research. Mesenchymal stem cells (MSCs) in particular have been extensively investigated as a potential therapeutic approach for cardiac regeneration, due to their distinctive characteristics. In this paper, we focus on the therapeutic applications of MSCs and their transition from the experimental benchside to the clinical bedside.

Vascular hypertrophy-associated hypertension of profilin1 transgenic mouse model leads to functional remodeling of peripheral arteries

Increased mechanical stress/hypertension in the vessel wall triggers the hypertrophic signaling pathway, resulting in structural remodeling of vasculature. Vascular hypertrophy of resistance vessels leads to reduced compliance and elevation of blood pressure. We showed before that increased expression of profilin1 protein in the medial layer of the aorta induces stress fiber formation, triggering the hypertrophic signaling resulting in vascular hypertrophy and, ultimately, hypertension in older mice. Our hypothesis is that profilin1 induced vascular hypertrophy in resistance vessels, which led to elevation of blood pressure, both of which contributed to the modulation of vascular function. Our results showed significant increases in the expression of alpha(1)- and beta(1)-integrins (280 + or - 6.3 and 325 + or - 7.4%, respectively) and the activation of the Rho/Rho-associated kinase (ROCK) II pathway (260 and 350%, respectively, P < 0.05) in profilin1 mesenteric arteries. The activation of Rho/ROCK led to the inhibition of endothelial nitric oxide synthase expression (39 + or - 5.4%; P < 0.05) and phosphorylation (35 + or - 4.5%; P < 0.05) but also an increase in myosin light chain 20 phosphorylation (372%, P < 0.05). There were also increases in hypertrophic signaling pathways in the mesenteric arteries of profilin1 mice such as phospho-extracellular signal-regulated kinase 1/2 and phospho-c-Jun NH(2)-terminal kinase (312.15 and 232.5%, respectively, P < 0.05). Functional analyses of mesenteric arteries toward the vasoactive drugs were assessed using wire-myograph and showed significant increases in the vascular responses of profilin1 mesenteric arteries toward phenylephrine, but significant decreases in response toward ROCK inhibitor Y-27632, ACh, sodium nitrite, and cytochalasin D. The changes in vascular responses in the mesenteric arteries of profilin1 mice are due to vascular hypertrophy and the elevation of blood pressure in the profilin1 transgenic mice.

Vascular remodeling-associated hypertension leads to left ventricular hypertrophy and contractile dysfunction in profilin-1 transgenic mice.

Abstract: Hypertension is a major health problem and a main risk factor for cardiovascular diseases. We have shown that overexpression of profilin-1 in blood vessels of transgenic mice generates mechanical tone and led to vascular remodeling/hypertension. However, little is known whether cardiac contractile performance in these mice is compromised. We investigated the in vivo contractile function and in vitro contractile performance using isolated papillary muscles from both right ventricle and left ventricle of profilin-1 mice at older age. Our results showed mild left ventricular hypertrophy and moderate systolic dysfunction in profilin-1 mice as evident by increased heart/body weight ratio and echocardiography analysis. Under near physiological conditions, right ventricle papillary muscles of profilin-1 mice maintained their peak isometric active developed tension, and the rate of force development over the entire frequency range of 4–14 Hz. Positive inotropic responses to increasing Ca2+ and &bgr;-adrenergic stimulation were also maintained. Conversely, left ventricular papillary muscles of profilin-1 mice exhibited depressed peak isometric, peak isometric active developed tension and rate of force development, and depressed positive inotropic responses to increasing Ca2+ and &bgr;-adrenergic stimulation. We here provide functional evidence that a significant contractile dysfunction in profilin-1 mice exists. Targeting vascular profilin-1 signaling could represent a promising therapeutic approach in hypertensive patients.

Rac-Induced Left Ventricular Dilation in Thyroxin-Treated ZmRacD Transgenic Mice: Role of Cardiomyocyte Apoptosis and Myocardial Fibrosis

The pathways inducing the critical transition from compensated hypertrophy to cardiac dilation and failure remain poorly understood. The goal of our study is to determine the role of Rac-induced signaling in this transition process. Our previous results showed that Thyroxin (T4) treatment resulted in increased myocardial Rac expression in wild-type mice and a higher level of expression in Zea maize RacD (ZmRacD) transgenic mice. Our current results showed that T4 treatment induced physiologic cardiac hypertrophy in wild-type mice, as demonstrated by echocardiography and histopathology analyses. This was associated with significant increases in myocardial Rac-GTP, superoxide and ERK1/2 activities. Conversely, echocardiography and histopathology analyses showed that T4 treatment induced dilated cardiomyopathy along with compensatory cardiac hypertrophy in ZmRacD mice. These were linked with further increases in myocardial Rac-GTP, superoxide and ERK1/2 activities. Additionally, there were significant increases in caspase-8 expression and caspase-3 activity. However, there was a significant decrease in p38-MAPK activity. Interestingly, inhibition of myocardial Rac-GTP activity and superoxide generation with pravastatin and carvedilol, respectively, attenuated all functional, structural, and molecular changes associated with the T4-induced cardiomyopathy in ZmRacD mice except the compensatory cardiac hypertrophy. Taken together, T4-induced ZmRacD is a novel mouse model of dilated cardiomyopathy that shares many characteristics with the human disease phenotype. To our knowledge, this is the first study to show graded Rac-mediated O2·− results in cardiac phenotype shift in-vivo. Moreover, Rac-mediated O2·− generation, cardiomyocyte apoptosis, and myocardial fibrosis seem to play a pivotal role in the transition from cardiac hypertrophy to cardiac dilation and failure. Targeting Rac signaling could represent valuable therapeutic strategy not only in saving the failing myocardium but also to prevent this transition process.

Cardiac remodeling caused by transgenic overexpression of a corn Rac gene

Rac1-GTPase activation plays a key role in the development and progression of cardiac remodeling. Therefore, we engineered a transgenic mouse model by overexpressing cDNA of a constitutively active form of Zea maize Rac gene (ZmRacD) specifically in the hearts of FVB/N mice. Echocardiography and MRI analyses showed cardiac hypertrophy in old transgenic mice, as evidenced by increased left ventricular (LV) mass and LV mass-to-body weight ratio, which are associated with relative ventricular chamber dilation and systolic dysfunction. LV hypertrophy in the hearts of old transgenic mice was further confirmed by an increased heart weight-to-body weight ratio and histopathology analysis. The cardiac remodeling in old transgenic mice was coupled with increased myocardial Rac-GTPase activity (372%) and ROS production (462%). There were also increases in α(1)-integrin (224%) and β(1)-integrin (240%) expression. This led to the activation of hypertrophic signaling pathways, e.g., ERK1/2 (295%) and JNK (223%). Pravastatin treatment led to inhibition of Rac-GTPase activity and integrin signaling. Interestingly, activation of ZmRacD expression with thyroxin led to cardiac dilation and systolic dysfunction in adult transgenic mice within 2 wk. In conclusion, this is the first study to show the conservation of Rho/Rac proteins between plant and animal kingdoms in vivo. Additionally, ZmRacD is a novel transgenic model that gradually develops a cardiac phenotype with aging. Furthermore, the shift from cardiac hypertrophy to dilated hearts via thyroxin treatment will provide us with an excellent system to study the temporal changes in cardiac signaling from adaptive to maladaptive hypertrophy and heart failure.

Role of Oxidative Stress in Thyroid Hormone-Induced Cardiomyocyte Hypertrophy and Associated Cardiac Dysfunction: An Undisclosed Story

Cardiac hypertrophy is the most documented cardiomyopathy following hyperthyroidism in experimental animals. Thyroid hormone-induced cardiac hypertrophy is described as a relative ventricular hypertrophy that encompasses the whole heart and is linked with contractile abnormalities in both right and left ventricles. The increase in oxidative stress that takes place in experimental hyperthyroidism proposes that reactive oxygen species are key players in the cardiomyopathy frequently reported in this endocrine disorder. The goal of this review is to shed light on the effects of thyroid hormones on the development of oxidative stress in the heart along with the subsequent cellular and molecular changes. In particular, we will review the role of thyroid hormone-induced oxidative stress in the development of cardiomyocyte hypertrophy and associated cardiac dysfunction, as well as the potential effectiveness of antioxidant treatments in attenuating these hyperthyroidism-induced abnormalities in experimental animal models.

Cardiomyocyte-specific overexpression of an active form of Rac predisposes the heart to increased myocardial stunning and ischemia-reperfusion injury

The GTP-binding protein Rac regulates diverse cellular functions including activation of NADPH oxidase, a major source of superoxide production (O(2)(·-)). Rac1-mediated NADPH oxidase activation is increased after myocardial infarction (MI) and heart failure both in animals and humans; however, the impact of increased myocardial Rac on impending ischemia-reperfusion (I/R) is unknown. A novel transgenic mouse model with cardiac-specific overexpression of constitutively active mutant form of Zea maize Rac D (ZmRacD) gene has been reported with increased myocardial Rac-GTPase activity and O(2)(·-) generation. The goal of the present study was to determine signaling pathways related to increased myocardial ZmRacD and to what extent hearts with increased ZmRacD proteins are susceptible to I/R injury. The effect of myocardial I/R was examined in young adult wild-type (WT) and ZmRacD transgenic (TG) mice. In vitro reversible myocardial I/R for postischemic cardiac function and in vivo regional myocardial I/R for MI were performed. Following 20-min global ischemia and 45-min reperfusion, postischemic cardiac contractile function and heart rate were significantly reduced in TG hearts compared with WT hearts. Importantly, acute regional myocardial I/R (30-min ischemia and 24-h reperfusion) caused significantly larger MI in TG mice compared with WT mice. Western blot analysis of cardiac homogenates revealed that increased myocardial ZmRacD gene expression is associated with concomitant increased levels of NADPH oxidase subunit gp91(phox), O(2)(·-), and P(21)-activated kinase. Thus these findings provide direct evidence that increased levels of active myocardial Rac renders the heart susceptible to increased postischemic contractile dysfunction and MI following acute I/R.

Differential involvement of various sources of reactive oxygen species in thyroxin-induced hemodynamic changes and contractile dysfunction of the heart and diaphragm muscles

Thyroid hormones are key regulators of basal metabolic state and oxidative metabolism. Hyperthyroidism has been reported to cause significant alterations in hemodynamics, and in cardiac and diaphragm muscle functions, all of which have been linked to increased oxidative stress. However, the definite source of increased reactive oxygen species (ROS) in each of these phenotypes is still unknown. The goal of the current study was to test the hypothesis that thyroxin (T4) may produce distinct hemodynamic, cardiac, and diaphragm muscle abnormalities by differentially affecting various sources of ROS. Wild-type and T4 mice with and without 2-week treatments with allopurinol (xanthine oxidase inhibitor), apocynin (NADPH oxidase inhibitor), L-NIO (nitric oxide synthase inhibitor), or MitoTEMPO (mitochondria-targeted antioxidant) were studied. Blood pressure and echocardiography were noninvasively evaluated, followed by ex vivo assessments of isolated heart and diaphragm muscle functions. Treatment with L-NIO attenuated the T4-induced hypertension in mice. However, apocynin improved the left-ventricular (LV) dysfunction without preventing the cardiac hypertrophy in these mice. Both allopurinol and MitoTEMPO reduced the T4-induced fatigability of the diaphragm muscles. In conclusion, we show here for the first time that T4 exerts differential effects on various sources of ROS to induce distinct cardiovascular and skeletal muscle phenotypes. Additionally, we find that T4-induced LV dysfunction is independent of cardiac hypertrophy and NADPH oxidase is a key player in this process. Furthermore, we prove the significance of both xanthine oxidase and mitochondrial ROS pathways in T4-induced fatigability of diaphragm muscles. Finally, we confirm the importance of the nitric oxide pathway in T4-induced hypertension.

The effect of selective antihypertensive drugs on the vascular remodeling-associated hypertension: insights from a profilin1 transgenic mouse model.

Hypertension represents a major risk factor for cardiovascular diseases. We have developed a novel transgenic mouse model by overexpressing the cDNA of human profilin1 in the blood vessels of transgenic mice, which led to vascular hypertrophy and hypertension. We assessed the effects of losartan, amlodipine, or atenolol on vascular hypertrophy-associated hypertension, by treating the profilin1 transgenic mice for 4 weeks. Our myograph results showed improvement in the contraction response toward phenylephrine and in the relaxation response toward acetylcholine and sodium nitrite in losartan- and amlodipine-treated profilin1 mice. Western blot analyses using mesenteric arteries of losartan- and amlodipine-treated profilin1 mice showed significant decreases in their signaling, respectively, as follows: the expression of α1 integrin (104% and 93%) and β1 integrin (116% and 109%); p-ERK1/2 (149% and 130%) and p-JNK (171% and 137%); the phospho-myosin light chain 20 (117% and 150%); and the ROCKII expression (125% and 180%). Conversely, there were significant increases in the endothelial nitric oxide synthase expression (82% and 80%) and activation (p-endothelial nitric oxide synthase) (78% and 76%). On the other hand, atenolol-treated profilin1 mice showed no significant change in all measured parameters. In conclusion, the profilin1 gene may represent a new therapeutic target in the treatment of vascular hypertrophy-associated hypertension.