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Objective Organ donation after circulatory death (DCD) is a potential solution for the shortage of suitable organs for transplant. Heart transplantation using DCD donors is not frequently performed due to the potential myocardial damage following warm ischemia. Heat shock protein (HSP) 90 has recently been investigated as a novel target to reduce ischemia/reperfusion injury. The objective of this study is to evaluate an innovative HSP90 inhibitor (HSP90i) as a cardioprotective agent in a model of DCD heart. Methods A DCD protocol was initiated in anesthetized Lewis rats by discontinuation of ventilation and confirmation of circulatory death by invasive monitoring. Following 15 minutes of warm ischemia, cardioplegia was perfused for 5 minutes at physiological pressure. DCD hearts were mounted on a Langendorff ex vivo heart perfusion system for reconditioning and functional assessment (60 minutes). HSP90i (0.01 μmol/L) or vehicle was perfused in the cardioplegia and during the first 10 minutes of ex vivo heart perfusion reperfusion. Following assessment, pro-survival pathway signaling was evaluated by western blot or polymerase chain reaction. Results Treatment with HSP90i preserved left ventricular contractility (maximum + dP/dt, 2385 ± 249 vs 1745 ± 150 mm Hg/s), relaxation (minimum –dP/dt, –1437 ± 97 vs 1125 ± 85 mm Hg/s), and developed pressure (60.7 ± 5.6 vs 43.9 ± 4.0 mm Hg), when compared with control DCD hearts (All P = .001). Treatment abrogates ischemic injury as demonstrated by a significant reduction of infarct size (2,3,5-triphenyl-tetrazolium chloride staining) of 7 ± 3% versus 19 ± 4% (P = .03), troponin T release, and mRNA expression of Bax/Bcl-2 (P Conclusions The cardioprotective effects of HSP90i when used following circulatory death might improve transplant organ availability by expanding the use of DCD hearts.

The increase in demand for cardiac transplantation throughout the years has fueled interest in donation after circulatory death (DCD) to expand the organ donor pool. However, the DCD process is associated with the risk of cardiac tissue injury due to the inevitable period of warm ischemia. Normothermic regional perfusion (NRP) allows for an in situ organ assessment, allowing the procurement of hearts determined to be viable. Here, we describe a clinically relevant large-animal model of DCD followed by NRP. Circulatory death is established in anesthetized pigs by stopping mechanical ventilation. After a preset warm ischemia period, an extracorporeal membrane oxygenator (ECMO) is used for a NRP period lasting at least 30 min. During this reperfusion period, the model allows the collection of various myocardial biopsies and blood samples for initial cardiac evaluation. Once NRP is weaned, biochemical, hemodynamic, and echocardiographic assessments of cardiac function and metabolism can be performed before organ procurement. This protocol closely simulates the clinical scenario previously described for DCD and NRP in heart transplantation and has the potential to facilitate studies aimed at decreasing ischemia-reperfusion injury and enhance cardiac functional preservation and recovery.

Despite several advances in medicine, ischaemic heart disease remains a major cause of morbidity and mortality. The unravelling of molecular mechanisms underlying disease pathophysiology has revealed targets for pharmacological interventions. However, transfer of these pharmcological possibilities to clinical use has been disappointing. Considering the complexity of ischaemic disease at the cellular and molecular levels, an equally multifaceted treatment approach may be envisioned. The pharmacological principle of ‘one target, one key’ may fall short in such contexts, and optimal treatment may involve one or many agents directed against complementary targets. Here, we introduce a ‘multi‐target approach to cardioprotection’ and propose heat shock protein 90 (HSP90) as a target of interest. We report on a member of a distinct class of HSP90 inhibitor possessing pleiotropic activity, which we found to exhibit potent infarct‐sparing effects.

The efficacy of cell therapy is limited by low retention and survival of transplanted cells in the target tissues. In this work, we hypothesize that pharmacological preconditioning with celastrol, a natural potent antioxidant, could improve the viability and functions of mesenchymal stromal cells (MSC) encapsulated within an injectable scaffold. Bone marrow MSCs from rat (rMSC) and human (hMSC) origin were preconditioned for 1 hour with celastrol 1 μM or vehicle (DMSO 0.1% v / v), then encapsulated within a chitosan-based thermosensitive hydrogel. Cell viability was compared by alamarBlue and live/dead assay. Paracrine function was studied first by quantifying the proangiogenic growth factors released, followed by assessing scratched HUVEC culture wound closure velocity and proliferation of HUVEC when cocultured with encapsulated hMSC. In vivo, the proangiogenic activity was studied by evaluating the neovessel density around the subcutaneously injected hydrogel after one week in rats. Preconditioning strongly enhanced the viability of rMSC and hMSC compared to vehicle-treated cells, with 90% and 75% survival versus 36% and 58% survival, respectively, after 7 days in complete media and 80% versus 64% survival for hMSC after 4 days in low serum media ( p < 0.05 ). Celastrol-treated cells increased quantities of proangiogenic cytokines compared to vehicle-pretreated cells, with a significant 3.0-fold and 1.8-fold increase of VEGFa and SDF-1α, respectively ( p < 0.05 ). The enhanced paracrine function of preconditioned MSC was demonstrated by accelerated growth and wound closure velocity of injured HUVEC monolayer ( p < 0.05 ) in vitro. Moreover, celastrol-treated cells, but not vehicle-treated cells, led to a significant increase of neovessel density in the peri-implant region after one week in vivo compared to the control (blank hydrogel). These results suggest that combining cell pretreatment with celastrol and encapsulation in hydrogel could potentiate MSC therapy for many diseases, benefiting particularly ischemic diseases.

Background: The shortage of organ donors is a major challenge in lung transplantation. To expand the lung donor pool, ex-vivo lung perfusion (EVLP) has emerged as a platform for assessment and reconditioning marginal donor lungs. In this study a stable and reproducible large animal model of lipopolysaccharide (LPS) induced systemic inflammation and acute lung injury (ALI) was developed.Methods: Pigs (n=6) were anesthetized and monitored. After infusion of LPS (20 μg/kg) for 1 hour, followed by a 90-minute response period, lungs were procured and kept on ice for 2 hours, followed by 4 hours of EVLP. Pulmonary function, inflammatory biomarkers and edema formation were measured in vivo before procurement and during EVLP. Pro and anti-inflammatory cytokines were assayed in blood and in EVLP perfusate, which were collected before and every 30 minutes after LPS administration and EVLP.Results: LPS infusion resulted in significant hemodynamic instability, characterized by marked pulmonary hypertension, decreased systemic blood pressure and increased heart rate. This was associated with increased levels of TNFα, IL-10, IL-6, but no change in IL-1β. Ex vivo assessment of injured lungs showed graft dysfunction characterized by impaired gas exchange and edema formation. The inflammatory profile showed stable but elevated TNFα levels, and continuous production of interleukins during EVLP.Conclusion: We describe a reproducible large animal model of LPS-induced systemic inflammation and ALI. EVLP alone was unable to recondition severely injured lungs. These findings suggest that the EVLP platform requires adjuncts such as targeted anti-inflammatory agents to allow reconditioning of marginal donor lungs.

Ischemia-reperfusion associated with prolonged warm ischemia during donation after circulatory death (DCD) induces acute lung injury. The objective of this study was to combine ex vivo lung perfusion (EVLP) and a heat shock protein-90 inhibitor (HSP90i) to recondition DCD organs and prevent primary graft dysfunction.Pigs (55 to 65 kg) were anesthetized, ventilated, and hemodynamically monitored. Cardiac arrest was induced with potassium chloride, and animals were left nonventilated for 2 hours. Lungs were procured and perfused in an EVLP platform for 4 hours by using a cellular perfusate. In the study group, the perfusate contained HSP90i and its transport vehicle (n = 4). In the control group, the perfusate contained only the transport vehicle (n = 4). Gas exchange, airway pressures, and compliance were measured. Pulmonary edema was assessed by bronchoscopy and weight measurement. Lung biopsy samples were obtained for histologic analyses and protein expression measurements.The use of HSP90i reduced lung weight gain to 8.4 ± 3.4% vs 26.6 ± 6.2% in the control group (P.05). There was reduced edema formation. The ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen at the end of EVLP was 423 ± 65 in the study group vs 339 ± 25 mm Hg in the control group, but this difference was not statistically significant. Lactate metabolism, pulmonary vascular resistance, and pulmonary arterial pressure improved during EVLP with the use of the HSP90i.The use of HSP90i with EVLP improves the lung reconditioning process. Further research is required to confirm whether these findings translate to benefit once transplanted and observed in vivo. Successful pharmacologic inhibitors may expand the donor pool in the context of DCD donors.

Cell therapy has the potential to improve healing of ischemic heart, repopulate injured myocardium and restore cardiac function. The tremendous hope and potential of stem cell therapy is well understood, yet recent trials involving cell therapy for cardiovascular diseases have yielded mixed results with inconsistent data thereby readdressing controversies and unresolved questions regarding stem cell efficacy for ischemic cardiac disease treatment. These controversies are believed to arise by the lack of uniformity of the clinical trial methodologies, uncertainty regarding the underlying reparative mechanisms of stem cells, questions concerning the most appropriate cell population to use, the proper delivery method and timing in relation to the moment of infarction, as well as the poor stem cell survival and engraftment especially in a diseased microenvironment which is collectively acknowledged as a major hindrance to any form of cell therapy. Indeed, the microenvironment of the failing heart exhibits pathological hypoxic, oxidative and inflammatory stressors impairing the survival of transplanted cells. Therefore, in order to observe any significant therapeutic benefit there is a need to increase resilience of stem cells to death in the transplant microenvironment while preserving or better yet improving their reparative functionality. Although stem cell differentiation into cardiomyocytes has been observed in some instance, the prevailing reparative benefits are afforded through paracrine mechanisms that promote angiogenesis, cell survival, transdifferentiate host cells and modulate immune responses. Therefore, to maximize their reparative functionality, ex vivo manipulation of stem cells through physical, genetic and pharmacological means have shown promise to enable cells to thrive in the post-ischemic transplant microenvironment. In the present work, we will overview the current status of stem cell therapy for ischemic heart disease, discuss the most recurring cell populations employed, the mechanisms by which stem cells deliver a therapeutic benefit and strategies that have been used to optimize and increase survival and functionality of stem cells including ex vivo preconditioning with drugs and a novel “pharmaco-optimizer” as well as genetic modifications.

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury. In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

Aims Cardiac ischemic conditioning has been shown to decrease ischemic injury in experimental models and clinically. Activation of survival pathways leading to heat shock proteins (HSP) modulation is an important contributor to this effect. We have previously shown that celastrol, an HSP90 modulator, achieves cardioprotection through activation of cytoprotective HSP's and heme-oxygenase-1 (HO-1). This is the first comparative evaluation of several modulators of HSP90 activity for cardioprotection. Furthermore, basic celastrol structure-activity relationship was characterized in order to develop novel potent infarct sparing agents suitable for clinical development. Main methods Combining in vitro cell culture using rat myocardial cell line exposed to ischemic and ischemia/reperfusion (I/R) stresses, and ex vivo Langendorff rat heart perfusion I/R model, we evaluated cardioprotective effects of various compounds. Selected signalling pathways were evaluated by western blot and reporter gene activation. Key findings From a variety of HSP90 modulator chemotypes, the celastrol family was most efficient in inducing cytoprotective HSP70 and HO-1 protein overexpression and cell survival in vitro. Celastrol and two synthetic analogs were protective against ischemia and prevented ischemia/reperfusion (I/R) injury when given as pre-treatment or at time of reperfusion, increasing viability and reducing mitochondrial permeability transition pore opening. Ex vivo experiments demonstrated that the two synthetic analogs show cardioprotective activity at lower concentrations compared to celastrol, with activation of multiple survival pathways. Significance Celastrol backbone is essential for cardioprotection through HSP90 activity modulation. These compounds hold promise as novel adjunct treatment to improve outcome in the clinical management of I/R injury.

The heart which has limited renewal and regenerative capacity is a prime target for cellular therapy. Stem cell transplantation has emerged as a promising therapeutic strategy to improve healing of the ischemic heart, repopulate the injured myocardium, and restore cardiac function. However, clinical usefulness is impacted by the quality and quantity of delivered cells, the suboptimal manipulations prior to transplantation, and the general poor viability of the cells transferred particularly to an ischemic microenvironment. Focus is now on developing new ways to enhance stem cell renewal and survival capacity before transplant. This can be done by physical, chemical, pharmacological, or genetic manipulation of cells followed by accurate evaluation of conditioning methods by validated tests.This chapter covers the proper handling of mesenchymal stem cells (human and rat lines) and methodologies to evaluate efficacy and the translational potential of conditioning methods. Specifically, we will cover stem cell culture methods, preconditioning protocols, viability assessment in hypoxic and oxidative challenges as encountered in an ischemic microenvironment, and the proliferative capacity of cells.

The heart which has limited renewal and regenerative capacity is a prime target for cellular therapy. Stem cell transplantation has emerged as a promising therapeutic strategy to improve healing of the ischemic heart, repopulate the injured myocardium, and restore cardiac function. However, clinical usefulness is impacted by the quality and quantity of delivered cells, the suboptimal manipulations prior to transplantation, and the general poor viability of the cells transferred particularly to an ischemic microenvironment. Focus is now on developing new ways to enhance stem cell renewal and survival capacity before transplant. This can be done by physical, chemical, pharmacological, or genetic manipulation of cells followed by accurate evaluation of conditioning methods by validated tests.This chapter covers the proper handling of mesenchymal stem cells (human and rat lines) and methodologies to evaluate efficacy and the translational potential of conditioning methods. Specifically, we will cover stem cell culture methods, preconditioning protocols, viability assessment in hypoxic and oxidative challenges as encountered in an ischemic microenvironment, and the proliferative capacity of cells.

Angiotensin converting enzyme 2 (ACE2) has been linked to cardiac dysfunction and hypertension-induced cardiac pathophysiology. In this study, we used a gene overexpression approach to investigate the role of ACE2 in cardiac function and remodeling after myocardial infarction. Rats received an intracardiac injection of 4.5×10 8 lentivirus containing ACE2 cDNA, followed by permanent coronary artery ligation (CAL) of the left anterior descending artery. At 24 hours and 6 weeks after surgery, cardiac functions, viability, and pathophysiology were assessed by MRI) and by histological analysis. At 24 hours post-CAL, left ventricular (LV) anterior wall motion was stunted to the same extent in control CAL and lenti-ACE2–treated CAL rats. However lenti-ACE2–treated CAL rats showed a 60% reduction in delayed contrast-enhanced LV volume after gadodiamide injection, indicating early ischemic protection of myocardium by ACE2. At 6 weeks after CAL, lenti-ACE2 rats demonstrated a complete rescue of cardiac output, a 41% rescue of ejection fraction, a 44% rescue in contractility, a 37% rescue in motion, and a 53% rescue in LV anterior (infracted) wall thinning compared with control CAL rats. No changes were observed in the LV posterior (noninfarcted) wall other than an 81% rescue in motion produced by ACE2 in CAL rats. Finally, infarct size measured by 2,3,5-triphenyl-tetrazolium chloride staining was not significantly different between the ligated groups. These observations demonstrate that cardiac overexpression of ACE2 exerts protective influence on the heart during myocardial infarction by preserving cardiac functions, LV wall motion and contractility, and by attenuating LV wall thinning.

Cardiac remodelling is a key risk factor for the development of heart failure in the chronic phase following myocardial infarction. Our previous studies have shown an anti-remodelling role of ACE2 (angiotensin-converting enzyme 2) in vivo during hypertension and that these protective effects are mediated through increased circulating levels of Ang-(1–7) [angiotensin-(1–7)]. In the present study, we have demonstrated that cardiac myocytes have modest ACE2 activity, whereas cardiac fibroblasts do not exhibit any endogenous activity. As fibroblasts are the major cell type found in an infarct zone following a myocardial infarction, we examined the effects of ACE2 gene delivery to cultured cardiac fibroblasts after acute hypoxic exposure. Cardiac fibroblasts from 5-day-old Sprague–Dawley rat hearts were grown to confluence and transduced with a lentiviral vector containing murine ACE2 cDNA under transcriptional control by the EF1α (elongation factor 1α) promoter (lenti-ACE2). Transduction of fibroblasts with lenti-ACE2 resulted in a viral dose-dependent increase in ACE2 activity. This was associated with a significant attenuation of both basal and hypoxia/re-oxygenation-induced collagen production by the fibroblasts. Cytokine production, specifically TGFβ (transforming growth factor β), by these cells was also significantly attenuated by ACE2 expression. Collectively, these results indicate that: (i) endogenous ACE2 activity is observed in cardiac myocytes, but not in cardiac fibroblasts; (ii) ACE2 overexpression in the cardiac fibroblast attenuates collagen production; and (iii) this prevention is probably mediated by decreased expression of cytokines. We conclude that ACE2 expression, limited to cardiac fibroblasts, may represent a novel paradigm for in vivo therapy following acute ischaemia.

Hypertension is one of the most important risk factors for cardiovascular diseases (CVDs). CVD extends across all populations and represents the leading cause of morbidity and mortality in industrialized countries. Hypertension increases the risk of peripheral arterial disease, cardiomyopathy, stroke, and renal failure. The disease affects &50 million Americans and goes undetected in at least one third of the population. In spite of the tremendous progress made by pharmacotherapy in the management and control of hypertension, there has been a steady escalation in its prevalence during the last decade. This has led many to conclude that traditional pharmacotherapy has reached an intellectual plateau and that novel and innovative approaches must be sought for the treatment, control, and possible cure of hypertension. As a result, efforts of our group and those of many others have been diverted to explore the use of gene transfer and gene therapy strategies for a long-term control of hypertension. We have argued that gene therapy offers potentially significant improvements and benefits over the use of traditional pharmacotherapy: (1) noncompliance by patients can be significantly reduced or even eliminated because of the fact that a single treatment involving gene transfer could remain effective for months or even years; (2) side effects associated with pharmacotherapy can be minimized as a result of specific targeting of a therapeutic gene in a given tissue and optimally influencing cardiovascular pathophysiology on an individual patient basis; and (3) with its potential to produce long-term beneficial outcomes in end-organ damage, the gene therapy strategy could lead to a cure of hypertension and its related pathophysiology. It is noteworthy to mention that current pharmacotherapies are often unable to reverse end-organ damage associated with hypertension. In order for the gene therapy for hypertension to be successful, one must provide conceptual support of its efficacy in …

Smooth muscle cell (SMC) apoptosis occurs at the onset of enalapril-induced regression of aortic hypertrophy in SHR. A potential mechanism is the correction of endothelial dysfunction (ED) leading to reduced production of reactive oxygen species and enhanced bioavailability of nitric oxide (NO), a potent apoptosis inducer. Stimulants of NO include the precursor L-arginine and the NO synthase cofactor tetrahydrobiopterin (BH(4)), which correct ED in several models. The objective was to examine the relationships between ED and the cell growth/death balance during vascular remodeling induced by enalapril in SHR. SHR, 10-week-old, received enalapril (ENA: 30 mg x kg(-1) x day(-1) p.o.) for 1 or 2 weeks, or a co-treatment of L-arginine (2.0 g x kg(-1) x day(-1) p.o.) and BH(4) (5.4 mg x kg(-1) x day(-1) i.p. twice daily) administered alone (group: LB) or in combination with enalapril (ENA+LB) for 1 week. Controls received vehicle. After 1 week, ED was completely corrected with LB but not affected significantly by ENA, whereas both treatments failed to induce SMC apoptosis or aortic remodeling. The correction of ED and the induction of SMC apoptosis (3.3-fold increase in TUNEL labeling) required 2 weeks of ENA treatment. The combination of LB with ENA for 1 week, however, was additive for the reduction of SMC proliferation, and synergistic for the induction of apoptosis and regression of vascular hypertrophy. These interactions were independent of blood pressure regulation. Our results suggest that the correction of ED is not sufficient to induce SMC apoptosis and vascular remodeling, although it facilitates these responses during enalapril treatment.

1. Treatment with enalapril induces smooth muscle cell apoptosis and regression of aortic hypertrophy in spontaneously hypertensive rats (SHRs), whereas combined blockade of angiotensin II AT(1) and AT(2) receptors does not. We postulated that vascular apoptosis with enalapril involves enhanced half-life of bradykinin (BK) and kinin B(2) receptor stimulation. 2. SHR, 11-weeks old, were treated for 4 weeks with enalapril (30 mg kg(-1) day(-1)), Hoe 140 (500 microg kg(-1) day(-1); B(2) receptor antagonist), alone or in combination. Controls received vehicle. 3. The half-life of hypotensive responses to intra-arterial bolus injections of BK were significantly increased in SHR anesthetized after 4 weeks of enalapril, an effect prevented by Hoe 140. The magnitude of BK-induced hypotension was significantly attenuated in all rats treated with Hoe 140. 4. As compared to placebo, enalapril treatment significantly reduced blood pressure (-34+/-2%), aortic hypertrophy (-20+/-3%), hyperplasia (-37+/-5%) and DNA synthesis (-61+/-8%), while it increased aortic DNA fragmentation by two-fold. Hoe 140 given alone or in combination with enalapril affected none of these parameters. 5. As a possible alternative mechanism, aortae isolated during the second week of enalapril treatment showed a transient upregulation of contractile responses to des-Arg(9)BK (EC(50)

Blockade of angiotensin type 1 (AT 1 ) receptors induces smooth muscle cell (SMC) death and regression of aortic hypertrophy in spontaneously hypertensive rats (SHR). We postulated that SMC death and vascular remodeling in this model may be attenuated by z-Val-Ala-Asp(OMe)-CH 2 F (z-VAD-fmk), a tripeptide inhibitor of caspase enzymes mediating apoptosis. To determine the time course of SMC death and aortic remodeling, SHR were treated with losartan (30 mg/kg per day) for up to 9.5 days. Transient SMC apoptosis occurred in the aortic media with a peak around day 5 of treatment, with increases in the Bax to Bcl-2 protein ratio (>3-fold), in active caspase-3 (5.6-fold), in TUNEL-positive nuclei (19-fold), preceding by 24 hours the peak activation of capase-9 (3.8-fold), and significant reductions in SMC number (46%) and aortic cross-sectional area (8.5%) at 5.5 days. The decrease in total aortic DNA reached significance at 6.5 days (29%). Blood pressure reduction with losartan was progressive and reached significance at day 7 of treatment. Next, we examined the causal link between vascular apoptosis and remodeling. SHR received placebo or losartan (30 mg/kg per day) for 6 days. During the last 24 hours, a subgroup of losartan-treated rats received 3 IV injections of z-VAD-fmk (cumulative dose: 4.4 mg · kg −1 ). All other rats received the vehicle, DMSO. The 24-hour cotreatment with z-VAD-fmk effectively prevented losartan-induced caspase-3 activation and internucleosomal DNA fragmentation, as well as SMC depletion and the reductions in aortic mass and DNA content. Together, these data suggest that caspase-dependent SMC death mediates the early phase of vascular remodeling in response to AT 1 receptor blockade in this model of hypertension.

[(3)H]-thymidine is commonly used to analyze the accumulation of [(3)H]-labeled chromatin fragments in cells undergoing apoptosis. This study shows that [(3)H]-thymidine incorporation within DNA is sufficient per se to inhibit growth and to induce apoptosis in canine kidney epithelial cells and porcine aorta endothelial cells. Despite high-level [(3)H]-thymidine-DNA labeling, rat vascular smooth muscle cells (VSMC) showed only modest inhibition of growth and induction of apoptosis compared to other cell types. Similarly to serum deprivation, apoptosis triggered by [(3)H]-thymidine labeling was sharply potentiated by VSMC transfection with a functional analogue of c-myc, E1A-adenoviral protein (VSMC-E1A), and was suppressed by stimulation of cAMP signaling with forskolin as well as by and Na/K pump inhibition with ouabain. Both apoptosis induction and growth suppression seen in [(3)H]-thymidine-treated VSMC-E1A were reduced by the pan-caspase inhibitor z-VAD.fmk. Thus, our results show that the differential efficiency of the apoptotic machinery determines cell type-specific attenuation of growth in cells with [(3)H]-thymidine-labeled DNA. They also demonstrate that [(3)H]-thymidine-treated and serum-deprived VSMC employ common intermediates of the apoptotic machinery, including steps that are potentiated by E1A-adenoviral protein and inhibited by activation of cAMP signaling as well as by inversion of the intracellular [Na(+)](i)/[K(+)](i) ratio.

Objective: Renin–angiotensin system inhibitors transiently induce apoptosis at the onset of cardiac hypertrophy regression in spontaneously hypertensive rats (SHRs). The focus of this study is to evaluate the cell selectivity of this response. Methods: SHRs were treated with valsartan or enalapril (30 mg kg−1 day−1) or placebo for 1 to 4 weeks. Stereological and morphological data were obtained from immunohistological analyses. Apoptosis was quantified by DEVDase (caspase-3-like) activity assay and immunoblot analysis of apoptosis-regulatory proteins (Bax and Bcl-2). Identification of the apoptotic cell type was conducted using in situ TUNEL labeling, in conjunction with α-sarcomeric actin or lectin immunoreactivity as markers for cardiomyocytes and endothelial cells, respectively. Results: Stereological analysis of the left ventricle revealed significant non-cardiomyocyte hyperplasia in placebo-treated SHRs (239±29×106 nuclei) as compared to untreated age-matched normotensive Wistar–Kyoto (WKY) rats (107±12×106). In contrast, the number of cardiomyocyte nuclei was comparable between untreated SHRs (48±4×106) and WKY rats. After 4 weeks of valsartan or enalapril treatment, SHRs showed significant reductions in systolic blood pressure (>28%), left ventricular hypertrophy (>9%) and cardiomyocyte cross-sectional area (>17%). Moreover, these treatments abolished non-cardiomyocyte hyperplasia in SHR left ventricle without affecting cardiomyocyte number, capillary density or number of capillary per cardiomyocyte nucleus. As a mechanism of cell deletion consistent with apoptosis induction, ventricles showed increased caspase-3 activation (>4.5-fold) as well as Bax to Bcl-2 protein ratio (>3.2-fold) within 2 weeks of valsartan or enalapril treatment. Immunohistological analysis revealed a significant increase in TUNEL-positive, lectin-negative non-cardiomyocytes, suggesting a rise in apoptotic interstitial fibroblasts in the left ventricle within 2 weeks of treatment with valsartan or enalapril (>63%), with a return to baseline (0.033±0.003%) at 4 weeks. Treatments did not affect right ventricular mass, apoptosis or cellularity. Conclusion: Cardiac apoptosis induction during regression of left ventricular hypertrophy reverses interstitial fibroblast hyperplasia in SHRs treated with inhibitors of the renin–angiotensin system.

Treatment with ouabain led to massive death of principal cells from collecting ducts (C7-MDCK), indicated by cell swelling, loss of mitochondrial function, an irregular pattern of DNA degradation, and insensitivity to pan-caspase inhibitor. Equimolar substitution of extracellular Na(+) by K(+) or choline(+) sharply attenuated the effect of ouabain on intracellular Na(+) and K(+) content but did not protect the cells from death in the presence of ouabain. In contrast to ouabain, inhibition of the Na(+)/K(+) pump in K(+)-free medium increased Na(+)(i) content but did not affect cell survival. In control and K(+)-free medium, ouabain triggered half-maximal cell death at concentrations of approximately 0.5 and 0.05 microM, respectively, which was consistent with elevation of Na(+)/K(+) pump sensitivity to ouabain in K(+)-depleted medium. Our results show for the first time that the death of ouabain-treated renal epithelial cells is independent of the inhibition of Na(+)/K(+) pump-mediated ion fluxes and the [Na(+)](i)]/[K(+)](i) ratio.

Objectives The IMPACT-CABG trial is the first North American multicenter phase II randomized study of intramyocardial delivery of autologous CD133 + stem cells in patients with chronic ischemic cardiomyopathy undergoing coronary artery bypass grafting. The primary objective was to demonstrate safety, including freedom from major adverse cardiac events. The secondary objective was to evaluate feasibility of same-day autologous cell preparation. Although the trial was not powered to evaluate LV function, exploratory data were collected. Methods After 7 open-label patients who received cells, patients randomly received stem cells or placebo (N = 40 total, 20 per center). After completion of coronary anastomoses, up to 10 million CD133 + , CD34 + , CD45 + triple-positive cells or placebo were injected into the infarct and border zones. Patients were followed up clinically and underwent magnetic resonance imaging preoperatively and after 6 months. Results There were no procedural complications from bone marrow isolation and cell injection, no in-hospital mortality, and no protocol-related complications. Four patients had transient renal insufficiency, with 1 death during 6-month follow-up. Magnetic resonance imaging revealed that left ventricular volumes and ejection fractions improved in all patients (no difference between groups). Conclusions The trial successfully met both primary and secondary objectives, demonstrating that same-day isolation and autologous CD133 + cell delivery with coronary artery bypass grafting is safe and feasible. The positive findings support a larger randomized, multicenter trial, with higher numbers of transplanted cells to demonstrate beneficial effects. The upcoming IMPACT-CABG II trial will evaluate higher cell doses and pharmacologic enhancement to determine whether these cells improve perfusion and myocardial function.

We previously reported that the AT1 receptor antagonist valsartan and the angiotensin converting enzyme (ACE) inhibitor enalapril decrease DNA synthesis and stimulate apoptosis in interstitial fibroblasts and epicardial mesothelial cells during regression of ventricular hypertrophy in spontaneously hypertensive rats (SHR). To examine the role of the AT2 receptor in this model, we studied hearts from SHR treated with valsartan or enalapril either alone or combined with the AT2 antagonist PD123319 for 1 or 2 weeks. Apoptosis was evaluated by quantification of DNA fragmentation or by TUNEL labeling. At 1 week, valsartan significantly increased ventricular DNA fragmentation, increased apoptosis in epicardial mesothelial cells, and decreased DNA synthesis. At 2 weeks, ventricular DNA content and cardiomyocyte cross-sectional area were significantly reduced. These valsartan-induced changes were attenuated by PD123319 co-administration. However, valsartan-induced increases in apoptosis of left ventricular interstitial non-cardiomyocytes was unaffected by the AT2 blocker. Enalapril-induced changes were similar to those observed with valsartan but were not affected by co-treatment with PD123319. These results demonstrate that AT1 and AT2 receptors act in a coordinated yet cell-specific manner to regulate cell growth and apoptosis in the left ventricle of SHR during AT1 receptor blockade but not ACE inhibition.

French Abstract: L’avenement des nouvelles technologies dans le milieu de la sante est souvent accueilli avec un certain scepticisme par la communaute medicale et ethico-juridique. En effet, la validite analytique et l’utilite clinique de ces outils ainsi que les consequences de leur utilisation sur l’analyse des responsabilites des medecins et autres professionnels de la sante demeurent sujets de debats. Une chose est claire : le medecin dans cette nouvelle ere fera face a plusieurs defis. Ceci dit, les nouvelles technologies ouvriront egalement la porte a plusieurs opportunites. Afin de discuter de ces points plus en profondeur, le Centre de genomique et politiques de l’Universite McGill a invite, le 30 mai dernier, des medecins, des juristes, des ethiciens, des conseillers en genetique ainsi que des membres des comites d’ethique a venir participer a un atelier intitule : « Le medecin face aux nouvelles technologies dans le domaine medical : nouvelles responsabilites? ». L’atelier avait deux objectifs precis : 1) familiariser les participants avec les obligations juridiques du medecin; 2) identifier et debattre des consequences de l’utilisation des nouvelles technologies sur les obligations juridiques du medecin. Trois cas pratiques composes de quatre grandes questions ont ete presentes aux participants. Chaque cas a couvert un domaine specifique, soit 1) le sequencage du genome entier; 2) les tests genetiques offerts directement aux consommateurs; et 3) les outils de detection rapide d’infections. Bien que l’accent ait ete mis sur le role du medecin, les questions ont eu pour but d’aborder egalement le role des divers acteurs dans le milieu de la recherche et de la pratique clinique. Ce rapport final exposera les resultats de ces echanges et les reponses des intervenants aux questions soumises. English Abstract: The advent of new technologies in the healthcare environment is often welcomed with some skepticism by the medical and ethical-legal community. The analytical validity and clinical utility of these tools, as well as the consequences of their use on the analysis of the responsibilities of physicians and other health professionals, remain debatable. One thing is clear: the physician in this new era will face several challenges. This being said, new technologies will also open the door to several opportunities. To address these issues in greater depth, the McGill Center of Genomics and Policy invited, on May 30th 2012, physicians, lawyers, ethicists, genetic counselors, and members of ethics committees, to attend a workshop entitled "The physician facing new technologies in the medical field: new responsibilities? ". The workshop had two specific objectives: 1) familiarize participants with the physician's legal obligations; 2) identify and discuss the consequences of the use of new technologies on the physician's legal obligations. Three practical cases with four main questions were presented to participants. Each case covered a specific domain, ie 1) whole-genome sequencing; (2) direct-to-consumer genetic tests; And 3) tools for rapid detection of infections. Although the emphasis was on the role of the physician, the questions were also intended to address the role of the various stakeholders in the research and clinical practice setting. This final report will present the results of these exchanges and the answers of the speakers to the questions submitted.

Hypertension afflicts over 65 million Americans and poses an increased risk for cardiovascular morbidity such as stroke, myocardial infarction and end-stage renal disease resulting in significant mortality. Overactivity of the renin-angiotensin system (RAS) has been identified as an important determinant that is implicated in the etiology of these diseases and therefore represents a major target for therapy. In spite of the successes of drugs inhibiting various elements of the RAS, the incidence of hypertension and cardiovascular diseases remain steadily on the rise. This has lead many investigators to seek novel and innovative approaches, taking advantage of new pathways and technologies, for the control and possibly the cure of hypertension and related pathologies. The main objective of this review is to forward the concept that gene therapy and the genetic targeting of the RAS is the future avenue for the successful control and treatment of hypertension and cardiovascular diseases. We will present argument that genetic targeting of angiotensin-converting enzyme 2 (ACE2), a newly discovered member of the RAS, is ideally poised for this purpose. This will be accomplished by discussion of the following: (i) summary of our current understanding of the RAS with a focus on the systemic versus tissue counterparts as they relate to hypertension and other cardiovascular pathologies; (ii) the newly discovered ACE2 enzyme with its physiological and pathophysiological implications; (iii) summary of the current antihypertensive pharmacotherapy and its limitations; (iv) the discovery and design of ACE inhibitors; (v) the emerging concepts for ACE2 drug design; (vi) the current status of genetic targeting of the RAS; (vii) the potential of ACE2 as a therapeutic target for hypertension and cardiovascular disease treatment; and (viii) future perspectives for the treatment of cardiovascular diseases.

Background Vasogen Inc.'s (Mississauga, Ontario, Canada) immune modulation therapy (IMT) is a therapy in which cells from the patient's own blood are modified by ex vivo exposure to specific physicochemical stressors, including oxidation, ultraviolet (UV) light, and an elevated temperature. The therapy has been shown to have a beneficial effect in models of inflammation and vascular diseases. This study tested the hypothesis that IMT can prevent renal ischemia-reperfusion (I/R) injury in rats. Methods Whole blood was collected from syngeneic age-matched donors by cardiac puncture. It was treated with a combination of controlled physiochemical stressors consisting of elevated temperature, a gas mixture of medical oxygen containing ozone, and UV light. The treated blood (150 microL) was injected in the gluteal muscle. Control animals received the same volume of untreated blood or physiological saline. Transient (45 or 60 minutes) left-renal ischemia was produced with simultaneous contralateral nephrectomy in treated and control spontaneously hypertensive rats (SHR). Young and old male and female rats were studied. Plasma creatinine, diuresis, and the survival rates of each group were compared. Renal apoptosis-necrosis was estimated by DNA laddering, histology, and in situ terminal deoxynucleotidyl transferase assay. mRNA levels of several regulators of apoptosis-regeneration were determined in control and postischemic kidneys by Northern blotting. Results IMT pretreatment of SHR significantly reduced renal I/R injury compared with equivalent placebo treatments consisting of untreated blood- or saline-injected SHR, as evidenced by a significant increase of the survival rate curves in young and old male SHR, which correlated with 24-hour postischemic diuresis. The increases in plasma creatinine following renal I/R were significantly lower in IMT-treated young male and old female SHR compared with saline or untreated blood-injected controls. Dilution analysis showed that the protective effect of treated blood was lost by dilution. Loss of epithelial cells was reduced in IMT-treated rats, with a significant decline in the peak of apoptosis 12 hours after acute ischemic renal injury. IMT did not modify the pattern of mRNA levels of several genes involved in the inflammation and regeneration processes. Conclusion Our data demonstrate that IMT prevents the destruction of kidney tissue and the resulting animal death caused by renal I/R injury.

Abstract —Angiotensin type 2 (AT 2 ) receptors for angiotensin II suppress cell growth and induce apoptosis in vitro, but their role is poorly defined in vivo. We reported that transient induction of smooth muscle cell (SMC) apoptosis precedes DNA synthesis inhibition and aortic hypertrophy regression in spontaneously hypertensive rats treated with the AT 1 antagonist losartan or the converting-enzyme inhibitor enalapril. Although both drugs are equipotent in reducing SMC number, apoptosis occurs significantly earlier with losartan than enalapril. To examine the role of AT 2 receptors in this model, spontaneously hypertensive rats were given valsartan, an AT 1 antagonist, or enalapril, in combination or not with the AT 2 antagonist PD123319 for 1 or 2 weeks. Control rats received vehicle. Systolic blood pressure was reduced similarly by valsartan and enalapril but it was not significantly affected by PD123319. Angiotensin II plasma levels were increased (6-fold) with valsartan and reduced (80%) with enalapril but unaffected by PD123319. Valsartan significantly increased internucleosomal DNA fragmentation indicative of apoptosis at 1 week only (2.7-fold) and significantly reduced aortic mass (18%), SMC number (33%), and DNA synthesis (24%, measured by 3 H-thymidine incorporation) at 2 weeks. These valsartan-induced changes were prevented by PD123319. In contrast, enalapril-induced DNA fragmentation (2-fold increase at 2 weeks) was not affected by PD123319. PD123319 given alone did not affect growth or apoptosis. AT 1 and AT 2 receptor mRNAs were detected in the aorta by reverse transcription–polymerase chain reaction. Together, these results provide the first evidence that AT 2 receptors mediate vascular mass regression by stimulating SMC apoptosis in vivo, an effect seen during AT 1 receptor blockade but not during converting-enzyme inhibition.