Shathiyah Kulandavelu

S‐Nitrosoglutathione Reductase Deficiency Enhances the Proliferative Expansion of Adult Heart Progenitors and Myocytes Post Myocardial Infarction

Background Mammalian heart regenerative activity is lost before adulthood but increases after cardiac injury. Cardiac repair mechanisms, which involve both endogenous cardiac stem cells (CSCs) and cardiomyocyte cell-cycle reentry, are inadequate to achieve full recovery after myocardial infarction (MI). Mice deficient in S-nitrosoglutathione reductase (GSNOR−⁄−), an enzyme regulating S-nitrosothiol turnover, have preserved cardiac function after MI. Here, we tested the hypothesis that GSNOR activity modulates cardiac cell proliferation in the post-MI adult heart. Methods and Results GSNOR−⁄− and C57Bl6/J (wild-type [WT]) mice were subjected to sham operation (n=3 GSNOR−⁄−; n=3 WT) or MI (n=41 GSNOR−⁄−; n=65 WT). Compared with WT,GSNOR−⁄− mice exhibited improved survival, cardiac performance, and architecture after MI, as demonstrated by higher ejection fraction (P<0.05), lower endocardial volumes (P<0.001), and diminished scar size (P<0.05). In addition, cardiomyocytes from post-MI GSNOR−⁄− hearts exhibited faster calcium decay and sarcomeric relaxation times (P<0.001). Immunophenotypic analysis illustrated that post-MI GSNOR−⁄− hearts demonstrated enhanced neovascularization (P<0.001), c-kit+ CSC abundance (P=0.013), and a ≈3-fold increase in proliferation of adult cardiomyocytes and c-kit+/CD45− CSCs (P<0.0001 and P=0.023, respectively) as measured by using 5-bromodeoxyuridine. Conclusions Loss of GSNOR confers enhanced post-MI cardiac regenerative activity, characterized by enhanced turnover of cardiomyocytes and CSCs. Endogenous denitrosylases exert an inhibitory effect over cardiac repair mechanisms and therefore represents a potential novel therapeutic target.

Regulation of oxygen delivery to the body via hypoxic vasodilation.

The respiratory system has traditionally been thought of as a two-gas model: hemoglobin (Hb) within red blood cells (RBC) binds oxygen in the lungs, delivers the oxygen to peripheral tissues, and binds carbon dioxide, which is returned to the lungs, released, and expired. However, fine-tuning of the system is required so that blood flow is preferentially shunted to tissues that have a greater need for oxygen. This mechanism, hypoxic vasodilation, is defined as the prompt vascular response to increased local demand for oxygen because of a change in metabolic activity in the absence of injury or disease (1, 2). This process involves detection of blood oxygen content by sensors and then a rapid transduction of the signal into a vasodilatory bioactivity (2). Emerging data suggest that Hb in the RBC not only functions as a vehicle to carry adequate amounts of oxygen to tissues, but also functions as an oxygen sensor and oxygen-responsive nitric oxide (NO) signal transducer, thereby regulating vascular tone (2, 3).

Alterations in β3-Adrenergic Cardiac Innervation and Nitric Oxide Signaling in Heart Failure⁎

Sympathetic activation of cardiac beta-adrenergic signaling pathways modulates inotropic, chronotropic, and lusitropic responses, providing fundamental control over cardiac reserve responses ([1][1]). Classically these effects are mediated through the 7-transmembrane spanning, G-protein coupled

Next-Generation Stem Cell Therapy: Genetically Modified Mesenchymal Stem Cells for Cardiac Repair

Although therapeutic advances have progressively reduced annual deaths from heart disease, cardiovascular disorders remain the leading cause of mortality and morbidity worldwide [1]. A main new therapeutic target is actual regeneration, fully repairing infarcted regions and restoring functioning myocardium following injury. In this regard, over the last ~15 years, the use of cell-based therapy has emerged as a leading approach to promoting myocardial regeneration and reducing myocardial infarct scar size. Avariety of stem cell populations including mesenchymal, cardiac and bone marrow-derived mononuclear cells have been identified and evaluated for their regenerative potential for the treatment of heart disease. One of the most favorable candidates for cellular therapy is the mesenchymal stem cell (MSC). There is substantial data from in vitro [2], preclinical [3–5] and clinical [6–9] studies supporting a multifactorial mechanism of action for the cardioreparative effects of MSCs. Main mechanisms include: reducing fibrosis and inflammation; stimulating angiogenesis; restoring contractile function and stimulating proliferation and activity of endogenous cardiomyocytes and cardiac stem cells. Although MSC therapy is promising, numerous challenges remain, including the source of donor cells, methods of cell delivery, and survival of transplanted cells in vivo. As such, enhancing the therapeutic effects of MSCs either by preconditioning with growth factors, hypoxia and/or drugs or through genetic modification is under active investigation. MSCs pre-conditioned by exposure to pro-angiogenic or anti-apoptotic growth factors [10, 11] such as hypoxia inducible factor-1 (HIF-1), vascular endothelial growth factor (VEGF), insulin-like growth factor [12], heme-oxygenase-1 or protein kinase B (Akt) show enhanced left ventricular (LV) function in animal models of MI. Similarly, stem cells transfected with angiopoietin-1, CXC chemokine receptor 4, PIM-1 kinase or stromalderived factor-1 showed enhanced engraftment and myocardial function, thereby preventing cardiac remodeling, in animal models of MI [10, 11, 13]. In this issue of Cardiovascular Drugs and Therapy, Chen et al. examined the therapeutic potential of MSCs overexpressing endothelial nitric oxide (NO) synthase (eNOS/NOS3) for the treatment of ischemic cardiac injury in rats [14]. NO produced by eNOS plays a broad range of regulatory roles in the cardiovascular system [15–17] including promotion of vasodilation, modulation of myocardial contractile responses, nitrosoredox imbalance, angiogenesis and inflammation. eNOSderived NO plays a cardioprotective role following MI as shown by eNOS knockout (KO) mice [18] which exhibited left ventricular (LV) dysfunction and enhanced interstitial fibrosis whereas cardiomyocyte-specific eNOS overexpressing mice [19] showed enhanced LV function and decreased myocyte hypertrophy following MI. Furthermore, local transfer of eNOS into ischemic rat hearts increased NO bioavailability, stimulated neovascularization, attenuated cardiac remodeling and suppressed oxidative stress associated apoptosis [20, 21]. Thus, eNOS may be an ideal candidate to enhance the cellular and therapeutic effects of MSCs. Chen et al. [14] showed that adenoviral delivery of the human eNOS gene into mouse bone marrow-derived MSCs (BM-MSCs) ameliorated the ischemic injury in rats. The combination of eNOS gene delivery and MSCs reduced infarct * Joshua M. Hare jhare@med.miami.edu

MP43-05 S-NITROSOGLUTATHIONE REDUCTASE (GSNOR) DEFICIENCY IS A NOVEL MODEL OF SECONDARY HYPOGONADISM

TUNEL, and immunohistochemical analysis for cleaved caspase-3 (apoptosis indicator), -8, -9, SHH and its receptor Patched. RESULTS: Cleaved caspase-3 was present in normal pelvic plexus and increased in a time dependent manner in all nerves of the pelvic plexus when the CN was crushed. Caspase 3 cleaved was identified primarily in glial cells, rather than neurons. Caspase 9 increased in glial cells of all nerves of the pelvic plexus, while very little caspase 8 was observed. SHH was abundant in neurons and glia of the CN, PN, HYG and ANC, while PTCH1 was identified only in neurons. CONCLUSIONS: Interruption of CN innervation, as occurs in the majority of prostatectomy patients, results in induction of apoptosis in other regions of the pelvic plexus, thus affecting continence. Apoptosis occurred primarily through the intrinsic pathway in response to CN injury. Identification of HYG and PN contribution to SUI, and involvement of the SHH pathway, identifies novel treatment avenues for intervention.

Alterations of tumor microenvironment by nitric oxide impedes castration-resistant prostate cancer growth

Significance This study presents insights into the underexplored areas of castration-resistant prostate cancer (CRPC) therapeutics—the role of nitric oxide (NO) in CRPC reduction through its microenvironment. Results of this study provide important information on the tumor reduction capabilities of increased NO levels and its mechanistic aspect and demonstrates the potential long-term efficacy of NO on CRPC. An in-depth understanding of how NO affects the tumor microenvironment will allow development of chemotherapeutics based on NO for a CRPC cure. Immune targeted therapy of nitric oxide (NO) synthases are being considered as a potential frontline therapeutic to treat patients diagnosed with locally advanced and metastatic prostate cancer. However, the role of NO in castration-resistant prostate cancer (CRPC) is controversial because NO can increase in nitrosative stress while simultaneously possessing antiinflammatory properties. Accordingly, we tested the hypothesis that increased NO will lead to tumor suppression of CRPC through tumor microenvironment. S-nitrosoglutathione (GSNO), an NO donor, decreased the tumor burden in murine model of CRPC by targeting tumors in a cell nonautonomous manner. GSNO inhibited both the abundance of antiinflammatory (M2) macrophages and expression of pERK, indicating that tumor-associated macrophages activity is influenced by NO. Additionally, GSNO decreased IL-34, indicating suppression of tumor-associated macrophage differentiation. Cytokine profiling of CRPC tumor grafts exposed to GSNO revealed a significant decrease in expression of G-CSF and M-CSF compared with grafts not exposed to GSNO. We verified the durability of NO on CRPC tumor suppression by using secondary xenograft murine models. This study validates the significance of NO on inhibition of CRPC tumors through tumor microenvironment (TME). These findings may facilitate the development of previously unidentified NO-based therapy for CRPC.

PD08-08 S-NITROSOGLUTATHIONE REDUCTASE (GSNOR) KNOCKOUT MICE: A NOVEL MODEL OF MALE INFERTILITY

model, Lecithin: Retinol Acyltransferase (LRAT) knockout (KO) mouse was used to answer our question. METHODS: 9 LRAT KO animals were fed either Vitamin A sufficient (ASuf) or Vitamin A deficient (ADef) diets for 8wks, with the latter known to produce SCO. H&E of testicular slides was used to assess histology. Immunofluorescence (IF) with antibodies against SYCP3 (marker of spermatocytes) was used to confirm loss of meiotic cells in LRAT KO Adef mice. RNAseq and smallRNA sequencing was performed using total RNA extracted from testes. Sequencing results were processed using JMP Genomics. Expression levels of GFRa1, PLZF, SCYP3, PRM1, TNP, CLU, and VIM were used to evaluate loss of different germ cell populations in the Adef group, and to normalize the data to the number of Sertoli cells. MicroRNA202-5p expression (Sertoli specific), miR-34c (germ cell specific) and let-7 (ubiquitous) were measured from sequencing data and further confirmed using QRT-PCR. Results were statistically significant at FRD1⁄40.01 RESULTS: 3 mice were evaluated in each respective group, LRAT Asuf and ADef conditions, at both 6 and 8wk time points. Histology, IF, and sequencing data demonstrated that spermatogenesis was present in all groups except for LRAT ADef mice at 8 weeks. Histology revealed heterogeneity, with most tubules resembling SCO in LRAT ADef testes. Normal spermatogenesis was observed in LRAT KO Asuf mice, and hypo spermatogenesis was observed in LRAT KO ADef mice at 6wks. There was no significant change in expression levels of miR202-5p between LRAT ASuf an ADef groups at 8wks. However, expression of miR34c was significantly decreased in the LRAT Adef group. Let-7 expression remained same. CONCLUSIONS: Loss of meiotic germ cells in LRAT KO ADef mice did not result in loss of miRNA-202-5p expression when compared to controls despite development of predominantly SCO histology. Our results provide strong evidence that the observed loss of expression of miRNA202-5p in men with SCO is not due to the primary loss of germ cells but is a result of primary miRNA-202-5p dysfunction in Sertoli cells.

Nitric Oxide Regulation of Cardiovascular Physiology and Pathophysiology

Abstract The nitric oxide (NO) signaling pathway participates in regulating numerous facets of cardiovascular function, including myocardial contractility, ventricular relaxation, mitochondrial respiration, and endothelial function. As such, this pathway contributes to numerous facets of cardiovascular pathophysiology. NO plays a main role in both regulating and responding to the redox state of the cell (nitroso-redox balance), which targets calcium handling, contractile mechanisms, vasoactive mechanisms, as well as stem cell aspects involved in cardiac regeneration. Thus, the interaction between NO and reactive oxygen species (ROS), the nitroso-redox signaling pathway, is critically important in cardiac physiology and pathophysiology and a fundamental therapeutic target. This chapter will address the cardiovascular implications of the biological balance between NO and ROS as they relate to cardiac physiology and the pathophysiology of heart disease and offer insights for the development of new therapeutic strategies.