Targeting stem cell niches and trafficking for cardiovascular therapy.

Regenerative cardiovascular medicine is the frontline of 21st-century health care. Cell therapy trials using bone marrow progenitor cells documented that the approach is feasible, safe and potentially beneficial in patients with ischemic disease. However, cardiovascular prevention and rehabilitation strategies should aim to conserve the pristine healing capacity of a healthy organism as well as reactivate it under disease conditions. This requires an increased understanding of stem cell microenvironment and trafficking mechanisms. Engagement and disengagement of stem cells of the osteoblastic niche is a dynamic process, finely tuned to allow low amounts of cells move out of the bone marrow and into the circulation on a regular basis. The balance is altered under stress situations, like tissue injury or ischemia, leading to remarkably increased cell egression. Individual populations of circulating progenitor cells could give rise to mature tissue cells (e.g. endothelial cells or cardiomyocytes), while the majority may differentiate to leukocytes, affecting the environment of homing sites in a paracrine way, e.g. promoting endothelial survival, proliferation and function, as well as attenuating or enhancing inflammation. This review focuses on the dynamics of the stem cell niche in healthy and disease conditions and on therapeutic means to direct stem cell/progenitor cell mobilization and recruitment into improved tissue repair.

Intermittent antegrade cardioplegia: isolated heart preservation with the Asporto heart preservation device

Background—A major problem in procurement of donor hearts is the limited time a donor heart remains viable. After cardiectomy, ischemic hypoxia is the main cause of donor heart degradation. The global myocardial ischemia causes a cascade of oxygen radical formation that cumulates in an elevation in hydrogen ions (decrease in pH), irreversible cellular injury, and potential microvascular changes in perfusion. Objective—To determine the changes of prolonged storage times on donor heart microvasculature and the effects of intermittent antegrade perfusion. Materials and Methods—Using porcine hearts flushed with a Ribosol-based cardioplegic solution, we examined how storage time affects microvascular myocardial perfusion by using contrast-enhanced magnetic resonance imaging at a mean (SD) of 6.1 (0.6) hours (n=13) or 15.6 (0.6) hours (n=11) after cardiectomy. Finally, to determine if administration of cardioplegic solution affects pH and microvascular perfusion, isolated hearts (group 1, n=9) given a single antegrade dose, were compared with hearts (group 2, n=8) given intermittent antegrade cardioplegia (150 mL, every 30 min, 150 mL/min) by a heart preservation device. Khuri pH probes in left and right ventricular tissue continuously measured hydrogen ion levels, and perfusion intensity on magnetic resonance images was plotted against time. Results—Myocardial perfusion measured via magnetic resonance imaging at 6.1 hours was significantly greater than at 15.6 hours (67% vs 30%, P= .00008). In group 1 hearts, the mean (SD) for pH at the end of 6 hours decreased to 6.2 (0.2). In group 2, hearts that received intermittent antegrade cardioplegia, pH at the end of 6 hours was higher at 6.7 (0.3) (P=.0005). Magnetic resonance imaging showed no significant differences between the 2 groups in contrast enhancement (group 1, 62%; group 2, 40%) or in the wet/dry weight ratio. Conclusion—Intermittent perfusion maintains a significantly higher myocardial pH than does a conventional single antegrade dose. This difference may translate into an improved quality of donor hearts procured for transplantation, allowing longer distance procurement, tissue matching, improved outcomes for transplant recipients, and ideally a decrease in transplant-related costs.

Gap junctions and connexin hemichannels in the regulation of haemostasis and thrombosis

Platelets are involved in the maintenance of haemostasis but their inappropriate activation leads to thrombosis, a principal trigger for heart attack and ischemic stroke. Although platelets circulate in isolation, upon activation they accumulate or aggregate together to form a thrombus, where they function in a coordinated manner to prevent loss of blood and control wound repair. Recent reports indicate that the stability and functions of a thrombus are maintained through sustained, contact dependent signalling between platelets. Given the role of gap junctions in the coordination of tissue responses, it was hypothesized that gap junctions may be present within a thrombus and mediate intercellular communication between platelets. Therefore studies were performed to explore the presence and functions of connexins in platelets. In this brief review, the roles of hemichannels and gap junctions in the control of thrombosis and haemostasis and the future directions for this research will be discussed.

Chagas disease-related mortality trends, state of Sao Paulo, Brazil, 1985 to 2006: a study using multiple causes of death

Objectives. To study mortality trends related to Chagas disease taking into account all mentions of this cause listed on any line or part of the death certificate. Methods. Mortality data for 1985-2006 were obtained from the multiple cause-of-death database maintained by the Sao Paulo State Data Analysis System (SEADE). Chagas disease was classified as the underlying cause-of-death or as an associated cause-of-death (non-underlying). The total number of times Chagas disease was mentioned on the death certificates was also considered. Results. During this 22-year period, there were 40 002 deaths related to Chagas disease: 34 917 (87.29%) classified as the underlying cause-of-death and 5 085 (12.71%) as an associated cause-of-death. The results show a 56.07% decline in the death rate due to Chagas disease as the underlying cause and a stabilized rate as associated cause. The number of deaths was 44.5% higher among men. The fact that 83.5% of the deaths occurred after 45 years of age reflects a cohort effect. The main causes associated with Chagas disease as the underlying cause-of-death were direct complications due to cardiac involvement, such as conduction disorders, arrhythmias and heart failure. Ischemic heart disease, cerebrovascular disorders and neoplasms were the main underlying causes when Chagas was an associated cause-of-death. Conclusions. For the total mentions to Chagas disease, a 51.34% decline in the death rate was observed, whereas the decline in the number of deaths was only 5.91%, being lower among women and showing a shift of deaths to older age brackets. Using the multiple cause-of-death method contributed to the understanding of the natural history of Chagas disease.

Subclinical thyroid dysfunctions are independent risk factors for mortality in a 7.5-year follow-up: the Japanese-Brazilian thyroid study

Objective: The currently available data concerning the influence of subclinical thyroid disease (STD) on morbidity and mortality are conflicting. Our objective was to investigate the relationships between STD and cardiometabolic profile and cardiovascular disease at baseline, as well as with all-cause and cardiovascular mortality in a 7.5-year follow-up. Design: Prospective, observational study. Methods: An overall of 1110 Japanese-Brazilians aged above 30 years, free of thyroid disease, and not taking thyroid medication at baseline were studied. In a cross-sectional analysis, we investigated the prevalence of STD and its relationship with cardiometabolic profile and cardiovascular disease. All-cause and cardiovascular mortality rates were assessed for participants followed for up to 7.5 years. Association between STD and mortality was drawn using multivariate analysis, adjusting for potential confounders. Results: A total of 913 (82.3%) participants had euthyroidism, 99 (8.7%) had subclinical hypothyroidism, and 69 (6.2%) had subclinical hyperthyroidism. At baseline, no association was found between STD and cardiometabolic profile or cardiovascular disease. Multivariate-adjusted hazard ratios (HRs (95% confidence interval)) for all-cause mortality were significantly higher for individuals with both subclinical hyperthyroidism (HR, 3.0 (1.5-5.9); n=14) and subclinical hypothyroidism (HR, 2.3 (1.2-4.4); n=13) than for euthyroid subjects. Cardiovascular mortality was significantly associated with subclinical hyperthyroidism (HR, 3.3 (1.4-7.5); n=8), but not with subclinical hypothyroidism (HR, 1.6 (0.6-4.2); n=5). Conclusion: In the Japanese-Brazilian population, subclinical hyperthyroidism is an independent risk factor for all-cause and cardiovascular mortality, while subclinical hypothyroidism is associated with all-cause mortality.

Long-range ozone transport and its impact on respiratory and cardiovascular health in the north of Portugal

Ozone dynamics depend on meteorological characteristics such as wind, radiation, sunshine, air temperature and precipitation. The aim of this study was to determine ozone trajectories along the northern coast of Portugal during the summer months of 2005, when there was a spate of forest fires in the region, evaluating their impact on respiratory and cardiovascular health in the greater metropolitan area of Porto. We investigated the following diseases, as coded in the ninth revision of the International Classification of Diseases: hypertensive disease (codes 401-405); ischemic heart disease (codes 410-414); other cardiac diseases, including heart failure (codes 426-428); chronic obstructive pulmonary disease and allied conditions, including bronchitis and asthma (codes 490-496); and pneumoconiosis and other lung diseases due to external agents (codes 500-507). We evaluated ozone data from air quality monitoring stations in the study area, together with data collected through HYbrid Single-Particle Lagrangian Integrated Trajectory (HYSPLIT) model analysis of air mass circulation and synoptic-scale zonal wind from National Centers for Environmental Prediction data. High ozone levels in rural areas were attributed to the dispersion of pollutants induced by local circulation, as well as by mesoscale and synoptic scale processes. The fires of 2005 increased the levels of pollutants resulting from the direct emission of gases and particles into the atmosphere, especially when there were incoming frontal systems. For the meteorological case studies analyzed, peaks in ozone concentration were positively associated with higher rates of hospital admissions for cardiovascular diseases, although there were no significant associations between ozone peaks and admissions for respiratory diseases.

Systemic delivery of adult stem cells improves cardiac function in spontaneously hypertensive rats

Heart regeneration after myocardial infarction (MI) can occur after cell therapy, but the mechanisms, cell types and delivery methods responsible for this improvement are still under investigation. In the present study, we evaluated the impact of systemic delivery of bone marrow cells (BMC) and cultivated mesenchymal stem cells (MSC) on cardiac morphology, function and mortality in spontaneously hypertensive rats (SHR) submitted to coronary occlusion. Female syngeneic adult SHR, submitted or not (control group; C) to MI, were treated with intravenous injection of MSC (MI + MSC) or BMC (MI + BM) from male rats and evaluated after 1, 15 and 30 days by echocardiography. Systolic blood pressure (SBP), functional capacity, histology, mortality rate and polymerase chain reaction for the Y chromosome were also analysed. Myocardial infarction induced a decrease in SBP and BMC, but not MSC, prevented this decrease. An improvement in functional capacity and ejection fraction (38 +/- 4, 39 +/- 3 and 58 +/- 2% for MI, MI + MSC and MI + BM, respectively; P < 0.05), as well as a reduction of the left ventricle infarcted area, were observed in rats from the MI + BM group compared with the other three groups. Treated animals had a significantly reduced lesion tissue score. The mortality rate in the C, MI + BM, MI + MSC and MI groups was 0, 0, 16.7 and 44.4%, respectively (P < 0.05 for the MI + MSC and MI groups compared with the C and MI + BM groups). The results of the present study suggest that systemic administration of BMC can improve left ventricular function, functional capacity and, consequently, reduce mortality in an animal model of MI associated with hypertension. We speculate that the cells transiently home to the myocardium, releasing paracrine factors that recruit host cells to repair the lesion.

Mobius Sequence in a Girl and Arthrogryposis in her Half-Brother: Distinct Phenotypes Caused By Prenatal Injuries

Mobius sequence is a congenital facial and abducens nerve palsy, frequently associated to abnormalities of extremities. Arthrogryposis multiplex congenital is defined as a congenital fixation of multiple joints seldom of neurogenic origin. Both sequences must have a genetic origin, but usually are sporadic cases related to environmental factors such as drugs exposition and maternal trauma. A 5-year-old girl and a 1-year-old boy were born with Mobius sequence and arthrogryposis multiplex congenital, respectively. During pregnancies, the mother had vaginal bleeding at 7 weeks and used crack (free-based cocaine) in the first trimester, respectively. The girl also has equinovarus talipes and autistic behavior. The boy has arthrogryposis with flexion contractures of the feet and knees. A vascular disruption, due to hemorrhage and cocaine exposure, causing a transient ischemic insult to embryos in a critical period of development may be responsible for distinct phenotypes in these cases.

Immobilized Kidney 28-kDa Endostatin- Related (KES28kDa) Fragment Promotes Endothelial Cell Survival

Background/Objective: Renal ischemia-hypoxia is a leading cause of acute kidney injury (AKI). Ischemia causes extracellular matrix breakdown of the tubular basement membrane. Endostatin (ES) is the C-terminal fragment of collagen XVIII generated by proteolytic cleavage. Recent studies have demonstrated that ES expression is upregulated in ischemic kidneys. The present study aimed to characterize ES from ischemic kidneys. Methods: Ischemic renal failure was induced via 45 min of occlusion of the left renal artery and vein. After the ischemic period, blood was collected. Kidneys were harvested and used for immunohistochemical testing and protein extraction. Three-step purification was used. Soluble and immobilized purified ES were tested in cell viability and adhesion assays. Results: The soluble KES28kDa inhibited endothelial cell proliferation: 25 versus 12.5 mu g (p < 0.05); 12.5 versus 3.15 mu g (p < 0.05). Immobilization of KES28kDa supports endothelial cell survival over the control p = 0.021). Human umbilical vein endothelial cells plated on immobilized KES28kDa showed an increase in membrane ruffles and stress fibers. Conclusion: These data demonstrate the local synthesis of a 28-kDa ES-related fragment following AKI and suggest its role in endothelium survival. Copyright (C) 2010 S. Karger AG, Basel

TUMOR NECROSIS FACTOR IS NOT ASSOCIATED WITH INTESTINAL ISCHEMIA/REPERFUSION-INDUCED LUNG INFLAMMATION

Intestinal ischemia-reperfusion (I/R) injury may cause acute systemic and lung inflammation. Here, we revisited the role of TNF-alpha in an intestinal I/R model in mice, showing that this cytokine is not required for the local and remote inflammatory response upon intestinal I/R injury using neutralizing TNF-alpha antibodies and TNF ligand-deficient mice. We demonstrate increased neutrophil recruitment in the lung as assessed by myeloperoxidase activity and augmented IL-6, granulocyte colony-stimulating factor, and KC levels, whereas TNF-alpha levels in serum were not increased and only minimally elevated in intestine and lung upon intestinal I/R injury. Importantly, TNF-alpha antibody neutralization neither diminished neutrophil recruitment nor any of the cytokines and chemokines evaluated. In addition, the inflammatory response was not abrogated in TNF and TNF receptors 1 and 2-deficient mice. However, in view of the damage on the intestinal barrier upon intestinal I/R with systemic bacterial translocation, we asked whether Toll-like receptor (TLR) activation is driving the inflammatory response. In fact, the inflammatory lung response is dramatically reduced in TLR2/4-deficient mice, confirming an important role of TLR receptor signaling causing the inflammatory lung response. In conclusion, endogenous TNF-alpha is not or minimally elevated and plays no role as a mediator for the inflammatory response upon ischemic tissue injury. By contrast, TLR2/4 signaling induces an orchestrated cytokine/chemokine response leading to local and remote pulmonary inflammation, and therefore disruption of TLR signaling may represent an alternative therapeutic target.

Bone marrow mononuclear cells attenuate fibrosis development after severe acute kidney injury

One of the early phases that lead to fibrosis progression is inflammation. Once this stage is resolved, fibrosis might be prevented. Bone marrow mononuclear cells (BMMCs) are emerging as a new therapy for several pathologies, including autoimmune diseases, because they enact immunosuppression. In this study we aimed to evaluate the role of BMMC administration in a model of kidney fibrosis induced by an acute injury. C57Bl6 mice were subjected to unilateral severe ischemia by clamping the left renal pedicle for 1 h. BMMCs were isolated from femurs and tibia, and after 6 h of reperfusion, 1 x 10(6) cells were administrated intraperitoneally. At 24 h after surgery, treated animals showed a significant decrease in creatinine and urea levels when compared with untreated animals. Different administration routes were tested. Moreover, interferon (IFN) receptor knockout BMMCs were used, as this receptor is necessary for BMMC activation. Labeled BMMCs were found in ischemic kidney on FACS analysis. This improved outcome was associated with modulation of inflammation in the kidney and systemic modulation, as determined by cytokine expression profiling. Despite non-amelioration of functional parameters, kidney mRNA expression of interleukin (IL)-6 at 6 weeks was lower in BMMC-treated animals, as were levels of collagen 1, connective tissue growth factor (CTGF), transforming growth factor-beta (TGF-beta) and vimentin. Protective molecules, such as IL-10, heme oxygenase 1 (HO-1) and bone morphogenetic 7 (BMP-7), were increased in treated animals after 6 weeks. Moreover, Masson and Picrosirius red staining analyses showed less fibrotic areas in the kidneys of treated animals. Thus, early modulation of inflammation by BMMCs after an ischemic injury leads to reduced fibrosis through modulation of early inflammation.

Administration of Neural Precursor Cells Ameliorates Renal Ischemia-Reperfusion Injury

In this study we evaluated whether administration of stem cells of neural origin (neural precursor cells, NPCs) could be protective against renal ischemia-reperfusion injury (IRI). We hypothesized that stem cell outcomes are not tissue-specific and that NPCs can improve tissue damage through paracrine mechanisms, especially due to immunomodulation. To this end, Wistar rats (200-250 g) were submitted to 1-hour ischemia and treated with NPCs (4 x 10(6) cells/animal) at 4 h of reperfusion. To serve as controls, ischemic animals were treated with cerebellum homogenate harvested from adult rat brain. All groups were sacrificed at 24 h of reperfusion. NPCs were isolated from rat fetus telencephalon and cultured until neurosphere formation (7 days). Before administration, NPCs were labeled with carboxyfluorescein diacetate succinimydylester (CFSE). Kidneys were harvested for analysis of cytokine profile and macrophage infiltration. At 24 h, NPC treatment resulted in a significant reduction in serum creatinine (IRI + NPC 1.21 + 0.18 vs. IRI 3.33 + 0.14 and IRI + cerebellum 2.95 + 0.78mg/dl, p < 0.05) and acute tubular necrosis (IRI + NPC 46.0 + 2.4% vs. IRI 79.7 + 14.2%, p < 0.05). NPC-CFSE and glial fibrillary acidic protein (GFAP)-positive cells (astrocyte marker) were found exclusively in renal parenchyma, which also presented GFAP and SOX-2 (an embryonic neural stem cell marker) mRNA expression. NPC treatment resulted in lower renal proinflammatory IL1-beta and TNF-alpha expression and higher anti-inflammatory IL-4 and IL-10 transcription. NPC-treated animals also had less macrophage infiltration and decreased serum proinflammatory cytokines (IL-1 beta, TNF-alpha and INF-gamma). Our data suggested that NPC therapy improved renal function by influencing immunological responses. Copyright (C) 2009 S. Karger AG, Basel

A Role for galectin-3 in renal tissue damage triggered by ischemia and reperfusion injury

Ischemic-reperfusion injury (IRI) triggers an inflammatory response involving neutrophils/macrophages, lymphocytes and endothelial cells. Galectin-3 is a multi-functional lectin with a broad range of action such as promotion of neutrophil adhesion, induction of oxidative stress, mastocyte migration and degranulation, and production of pro-inflammatory cytokines. The aim of this study was evaluate the role of galectin-3 in the inflammation triggered by IRI. Galectin-3 knockout (KO) and wild type (wt) mice were subjected to 45 min of renal pedicle occlusion. Blood and kidney samples were collected at 6, 24, 48 and 120 h. Blood urea was analyzed enzymatically, while MCP-1, IL-6 and IL-1 beta were studied by real-time PCR. Reactive oxygen species (ROS) was investigated by flow cytometry. Morphometric analyses were performed at 6, 24, 48 and 120 h after reperfusion. Urea peaked at 24 h, being significantly lower in knockout animals (wt = 264.4 +/- 85.21 mg/dl vs. gal-3 KO = 123.74 +/- 29.64 mg/dl, P = 0.001). Galectin-3 knockout animals presented less acute tubular necrosis and a more prominent tubular regeneration when compared with controls concurrently with lower expression of MCP-1, IL-6, IL-1 beta, less macrophage infiltration and lower ROS production at early time points. Galectin-3 seems to play a role in renal IRI involving the secretion of macrophage-related chemokine, pro-inflammatory cytokines and ROS production.

Neonatal mitochondrial encephaloneuromyopathy due to a defect of mitochondrial protein synthesis

Mitochondrial diseases are clinically and genetically heterogeneous disorders due to primary mutations in mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). We studied a male infant with severe congenital encephalopathy, peripheral neuropathy, and myopathy. The patient`s lactic acidosis and biochemical defects of respiratory chain complexes I, III, and IV in muscle indicated that he had a mitochondrial disorder while parental consanguinity suggested autosomal recessive inheritance. Cultured fibroblasts from the patient showed a generalized defect of mitochondrial protein synthesis. Fusion of cells from the patient with 143B206 rho(0) cells devoid of mtDNA restored cytochrome c oxidase activity confirming the nDNA origin of the disease. Our studies indicate that the patient has a novel autosomal recessive defect of mitochondrial protein synthesis. (C) 2008 Elsevier B.V. All rights reserved.

Renal ischemia in rats: Mitochondria function and laser autofluorescence

Ischemia-reperfusion injury is the major cause of organ dysfunction or even nonfunction following transplantation. It can attenuate the long-term survival of transplanted organs. To evaluate the severity of renal ischemia injury determined by histology, we applied laser(442 nm and 532 nm) induced fluorescence (LIF), mitochondria respiration, and membrane swelling to evaluate 28 Wistar rats that underwent left kidney warm ischemia for 20, 40, 60, or 80 minutes. LIF performed before ischemia (control) was repeated at 20, 40, 60, and 80 minutes thereafter. We harvested left kidney tissue samples immediately after LIF determination for histology and mitochondrial analyses: state 3 and 4 respiration, respiration control rate (RCR), and membrane swelling. The association of optic spectroscopy with histological damage showed: LIF, 442 nm (r(2) = 0.39, P < .001) and 532 nm, (r(2) = 0.18, P = .003); reflecting laser/fluorescence-induced, 442 nm (r(2) = 0.20, P = .002) and 532 nm (r(2) = 0.004, P = .67). The associations between mitochondria function and tissue damage were: state 3 respiration (r(2) = 0.43, P = .0004), state 4 respiration (r(2) = 0.03, P = 0.38), RCR (r(2) = 0.28, P = .007), and membrane swelling (r(2) = 0.02, P = .43). The intensity of fluorescence emitted by tissue excited by laser, especially at a wave length of 442 nm, was determined in real time. Mitochondrial state 3 respiration and respiratory control ratio also exhibited good correlations with the grade of ischemic tissue damage.