Aldose Reductase (AR) Mediates and Perivascular Adipose Tissue (PVAT) Modulates Endothelial Dysfunction of Short-Term High-Fat Diet Feeding in Mice.

Increased adiposity of both visceral and perivascular adipose tissue (PVAT) depots is associated with an increased risk of diabetes and cardiovascular disease (CVD). Under healthy conditions, PVAT modulates vascular tone via the release of PVAT-derived relaxing factors, including adiponectin and leptin. However, when PVAT expands with high-fat diet (HFD) feeding, it appears to contribute to the development of endothelial dysfunction (ED). Yet, the mechanisms by which PVAT alters vascular health are unclear. Aldose reductase (AR) catalyzes glucose reduction in the first step of the polyol pathway and has been long implicated in diabetic complications including neuropathy, retinopathy, nephropathy, and vascular diseases. To better understand the roles of both PVAT and AR in HFD-induced ED, we studied structural and functional changes in aortic PVAT induced by short-term HFD (60% kcal fat) feeding in wild type (WT) and aldose reductase-null (AR-null) mice. Although 4 weeks of HFD feeding significantly increased body fat and PVAT mass in both WT and AR-null mice, HFD feeding induced ED in the aortas of WT mice but not of AR-null mice. Moreover, HFD feeding augmented endothelial-dependent relaxation in aortas with intact PVAT only in WT and not in AR-null mice. These data indicate that AR mediates ED associated with short-term HFD feeding and that ED appears to provoke 'compensatory changes' in PVAT induced by HFD. As these data support that the ED of HFD feeding is AR-dependent, vascular-localized AR remains a potential target of temporally selective intervention.

Enhanced beta-1 adrenergic receptor responsiveness in coronary arterioles following intravenous stromal vascular fraction therapy in aged rats.

Our past study showed that a single tail vein injection of adipose-derived stromal vascular fraction (SVF) into old rats was associated with improved dobutamine-mediated coronary flow reserve. We hypothesize that i.v. injection of SVF improves coronary microvascular function in aged rats via alterations in beta adrenergic microvascular signaling. Female Fischer-344 rats aged young (3 months, n=32) and old (24 months, n=30) were utilized, along with two cell therapies intravenously injected in old rats four weeks prior to sacrifice: 1x107 green fluorescent protein (GFP+) SVF cells (O+SVF, n=21), and 5x106 GFP+ bone-marrow mesenchymal stromal cells (O+BM, n=6), both harvested from young donors. Cardiac ultrasound and pressure-volume measurements were obtained, and coronary arterioles were isolated from each group for microvessel reactivity studies and immunofluorescence staining. Coronary flow reserve decreased with advancing age, but this effect was rescued by the SVF treatment in the O+SVF group. Echocardiography showed an age-related diastolic dysfunction that was improved with SVF to a greater extent than with BM treatment. Coronary arterioles isolated from SVF-treated rats showed amelioration of the age-related decrease in vasodilation to a non-selective ß-AR agonist. I.v. injected SVF cells improved ß-adrenergic receptor-dependent coronary flow and microvascular function in a model of advanced age.

Chronic optical pacing conditioning of h-iPSC engineered cardiac tissues.

The immaturity of human induced pluripotent stem cell derived engineered cardiac tissues limits their ability to regenerate damaged myocardium and to serve as robust in vitro models for human disease and drug toxicity studies. Several chronic biomimetic conditioning protocols, including mechanical stretch, perfusion, and/or electrical stimulation promote engineered cardiac tissue maturation but have significant technical limitations. Non-contacting chronic optical stimulation using heterologously expressed channelrhodopsin light-gated ion channels, termed optogenetics, may be an advantageous alternative to chronic invasive electrical stimulation for engineered cardiac tissue conditioning. We designed proof-of-principle experiments to successfully transfect human induced pluripotent stem cell derived engineered cardiac tissues with a desensitization resistant, chimeric channelrhodopsin protein, and then optically paced engineered cardiac tissues to accelerate maturation. We transfected human induced pluripotent stem cell engineered cardiac tissues using an adeno-associated virus packaged chimeric channelrhodopsin and then verified optically paced by whole cell patch clamp. Engineered cardiac tissues were then chronically optically paced above their intrinsic beat rates in vitro from day 7 to 14. Chronically optically paced resulted in improved engineered cardiac tissue electrophysiological properties and subtle changes in the expression of some cardiac relevant genes, though active force generation and histology were unchanged. These results validate the feasibility of a novel chronically optically paced paradigm to explore non-invasive and scalable optically paced-induced engineered cardiac tissue maturation strategies.

Adipose-derived cells improve left ventricular diastolic function and increase microvascular perfusion in advanced age.

An early manifestation of coronary artery disease in advanced age is the development of microvascular dysfunction leading to deficits in diastolic function. Our lab has previously shown that epicardial treatment with adipose-derived stromal vascular fraction (SVF) preserves microvascular function following coronary ischemia in a young rodent model. Follow-up studies showed intravenous (i.v.) delivery of SVF allows the cells to migrate to the walls of small vessels and reset vasomotor tone. Therefore we tested the hypothesis that the i.v. cell injection of SVF would reverse the coronary microvascular dysfunction associated with aging in a rodent model. Fischer 344 rats were divided into 4 groups: young control (YC), old control (OC), old + rat aortic endothelial cells (O+EC) and old + GFP+ SVF cells (O+SVF). After four weeks, cardiac function and coronary flow reserve (CFR) were measured via echocardiography, and hearts were explanted either for histology or isolation of coronary arterioles for vessel reactivity studies. In a subgroup of animals, microspheres were injected during resting and dobutamine-stimulated conditions to measure coronary blood flow. GFP+ SVF cells engrafted and persisted in the myocardium and coronary vasculature four weeks following i.v. injection. Echocardiography showed age-related diastolic dysfunction without accompanying systolic dysfunction; diastolic function was improved in old rats after SVF treatment. Ultrasound and microsphere data both showed increased stimulated coronary blood flow in O+SVF rats compared to OC and O+EC, while isolated vessel reactivity was mostly unchanged. I.v.-injected SVF cells were capable of incorporating into the vasculature of the aging heart and are shown in this study to improve CFR and diastolic function in a model of advanced age. Importantly, SVF injection did not lead to arrhythmias or increased mortality in aged rats. SVF cells provide an autologous cell therapy option for treatment of microvascular and cardiac dysfunction in aged populations.

Neonatal Murine Engineered Cardiac Tissue Toxicology Model: Impact of Metallothionein Overexpression on Cadmium-Induced Injury.

Engineered cardiac tissues (ECTs) serve as robust in vitro models to study human cardiac diseases including cardiac toxicity assays due to rapid structural and functional maturation and the ability to vary ECT composition. Metallothionein (MT) has been shown to be cardioprotective for environmental toxicants including heavy metals. To date, studies on the role of cardiomyocyte (CM)-specific MT expression and function have occurred in dissociated single cell assays or expensive in vivo small animal models. Therefore, we generated 3D ECTs using neonatal mouse ventricular cells from wild-type (WT) and the CM-specific overexpressing MT-transgenic (MT-TG) to determine the effect of MT overexpression on ECT maturation and function. Because Cadmium (Cd) is an environmentally prevalent heavy metal toxicant with direct negative impact on cardiac structure and function, we then determined the effect of MT overexpression to reduce Cd mediated CM toxicity within ECTs. We found: (1) structural and functional maturation was similar in WT and MT-TG ECTs; (2) Cd exposure negatively impacted ECT cell survival, maturation, and function; and (3) MT-ECTs showed reduced Cd toxicity as defined by reduced cleaved caspase 3, reduced Bax/Bcl2 ratio, reduced TdT-mediated dUTP nick-end labeling positive cells, reduced CM loss after Cd treatment, and delayed onset of cardiac dysfunction after Cd treatment. Thus, neonatal murine ECTs can serve as a robust in vitro model for heavy metal toxicity screening and as a platform to evaluate the role cardioprotective mechanisms, such as the MT-TG model, on environmentally relevant toxicants.

Quantification of Cardiomyocyte Alignment from Three-Dimensional (3D) Confocal Microscopy of Engineered Tissue.

Biological tissues have complex, three-dimensional (3D) organizations of cells and matrix factors that provide the architecture necessary to meet morphogenic and functional demands. Disordered cell alignment is associated with congenital heart disease, cardiomyopathy, and neurodegenerative diseases and repairing or replacing these tissues using engineered constructs may improve regenerative capacity. However, optimizing cell alignment within engineered tissues requires quantitative 3D data on cell orientations and both efficient and validated processing algorithms. We developed an automated method to measure local 3D orientations based on structure tensor analysis and incorporated an adaptive subregion size to account for multiple scales. Our method calculates the statistical concentration parameter, κ, to quantify alignment, as well as the traditional orientational order parameter. We validated our method using synthetic images and accurately measured principal axis and concentration. We then applied our method to confocal stacks of cleared, whole-mount engineered cardiac tissues generated from human-induced pluripotent stem cells or embryonic chick cardiac cells and quantified cardiomyocyte alignment. We found significant differences in alignment based on cellular composition and tissue geometry. These results from our synthetic images and confocal data demonstrate the efficiency and accuracy of our method to measure alignment in 3D tissues.

Impact of Cell Composition and Geometry on Human Induced Pluripotent Stem Cells-Derived Engineered Cardiac Tissue.

The current study describes a scalable, porous large-format engineered cardiac tissue (LF-ECT) composed of human induced pluripotent stem cells (hiPSCs) derived multiple lineage cardiac cells with varied 3D geometries and cell densities developed towards the goal of scale-up for large animal pre-clinical studies. We explored multiple 15 × 15 mm ECT geometries using molds with rectangular internal staggered posts (mesh, ME), without posts (plain sheet, PS), or long parallel posts (multiple linear bundles, ML) and a gel matrix containing hiPSC-derived cardiomyocytes, endothelial, and vascular mural cells matured in vitro for 14 days. ME-ECTs displayed the lowest dead cell ratio (p < 0.001) and matured into 0.5 mm diameter myofiber bundles with greater 3D cell alignment and higher active stress than PS-ECTs. Increased initial ECT cell number beyond 6 M per construct resulted in reduced cell survival and lower active stress. The 6M-ME-ECTs implanted onto 1 week post-infarct immune tolerant rat hearts engrafted, displayed evidence for host vascular coupling, and recovered myocardial structure and function with reduced scar area. We generated a larger (30 × 30 mm) ME-ECT to confirm scalability. Thus, large-format ECTs generated from hiPSC-derived cardiac cells may be feasible for large animal preclinical cardiac regeneration paradigms.

The myocardial regenerative potential of three-dimensional engineered cardiac tissues composed of multiple human iPS cell-derived cardiovascular cell lineages.

Human induced pluripotent stem cells (hiPSCs) are a robust source for cardiac regenerative therapy due to their potential to support autologous and allogeneic transplant paradigms. The in vitro generation of three-dimensional myocardial tissue constructs using biomaterials as an implantable hiPSC-derived myocardium provides a path to realize sustainable myocardial regeneration. We generated engineered cardiac tissues (ECTs) from three cellular compositions of cardiomyocytes (CMs), endothelial cells (ECs), and vascular mural cells (MCs) differentiated from hiPSCs. We then determined the impact of cell composition on ECT structural and functional properties. In vitro force measurement showed that CM+EC+MC ECTs possessed preferential electromechanical properties versus ECTs without vascular cells indicating that incorporation of vascular cells augmented tissue maturation and function. The inclusion of MCs facilitated more mature CM sarcomeric structure, preferential alignment, and activated multiple tissue maturation pathways. The CM+EC+MC ECTs implanted onto infarcted, immune tolerant rat hearts engrafted, displayed both host and graft-derived vasculature, and ameliorated myocardial dysfunction. Thus, a composition of CMs and multiple vascular lineages derived from hiPSCs and incorporated into ECTs promotes functional maturation and demonstrates myocardial replacement and perfusion relevant for clinical translation.

Feasibility study of particulate extracellular matrix (P-ECM) and left ventricular assist device (HVAD) therapy in chronic ischemic heart failure bovine model.

Myocardial recovery with left ventricular assist device (LVAD) support is uncommon and unpredictable. We tested the hypothesis that injectable particulate extracellular matrix (P-ECM) with LVAD support promotes cell proliferation and improves cardiac function. LVAD, P-ECM, and P-ECM + LVAD therapies were investigated in chronic ischemic heart failure (IHF) calves induced using coronary embolization. Particulate extracellular matrix emulsion (CorMatrix, Roswell, GA) was injected intramyocardially using a 7 needle pneumatic delivery tool. Left ventricular assist devices (HVAD, HeartWare) were implanted in a left ventricle (LV) apex to proximal descending aorta configuration. Cell proliferation was identified using BrdU (5 mg/kg) injections over the last 45 treatment days. Echocardiography was performed weekly. End-organ regional blood flow (RBF) was quantified at study endpoints using fluorescently labeled microspheres. Before treatment, IHF calves had an ejection fraction (EF) of 33 ± 2% and left ventricular end-diastolic volume of 214 ± 18 ml with cardiac cachexia (0.69 ± 0.06 kg/day). Healthy weight gain was restored in all groups (0.89 ± 0.03 kg/day). EF increased with P-ECM + HVAD from 36 ± 5% to 75 ± 2%, HVAD 38 ± 4% to 58 ± 5%, and P-ECM 27 ± 1% to 66 ± 6%. P-ECM + HVAD demonstrated the largest increase in cell proliferation and end-organ RBF. This study demonstrates the feasibility of combined LVAD support with P-ECM injection to stimulate new cell proliferation and improve cardiac function, which warrants further investigation.

Effects of physiologic mechanical stimulation on embryonic chick cardiomyocytes using a microfluidic cardiac cell culture model.

Hemodynamic mechanical cues play a critical role in the early development and functional maturation of cardiomyocytes (CM). Therefore, tissue engineering approaches that incorporate immature CM into functional cardiac tissues capable of recovering or replacing damaged cardiac muscle require physiologically relevant environments to provide the appropriate mechanical cues. The goal of this work is to better understand the subcellular responses of immature cardiomyocytes using an in vitro cardiac cell culture model that realistically mimics in vivo mechanical conditions, including cyclical fluid flows, chamber pressures, and tissue strains that could be experienced by implanted cardiac tissues. Cardiomyocytes were cultured in a novel microfluidic cardiac cell culture model (CCCM) to achieve accurate replication of the mechanical cues experienced by ventricular CM. Day 10 chick embryonic ventricular CM (3.5 × 10(4) cell clusters per cell chamber) were cultured for 4 days in the CCCM under cyclic mechanical stimulation (10 mmHg, 8-15% stretch, 2 Hz frequency) and ventricular cells from the same embryo were cultured in a static condition for 4 days as controls. Additionally, ventricular cell suspensions and ventricular tissue from day 16 chick embryo were collected and analyzed for comparison with CCCM cultured CM. The gene expressions and protein synthesis of calcium handling proteins decreased significantly during the isolation process. Mechanical stimulation of the cultured CM using the CCCM resulted in an augmentation of gene expression and protein synthesis of calcium handling proteins compared to the 2D constructs cultured in the static conditions. Further, the CCCM conditioned 2D constructs have a higher beat rate and contractility response to isoproterenol. These results demonstrate that early mechanical stimulation of embryonic cardiac tissue is necessary for tissue proliferation and for protein synthesis of the calcium handling constituents required for tissue contractility. Thus, physiologic mechanical conditioning may be essential for generating functional cardiac patches for replacement of injured cardiac tissue.

Gene expression profiles in engineered cardiac tissues respond to mechanical loading and inhibition of tyrosine kinases.

Engineered cardiac tissues (ECTs) are platforms to investigate cardiomyocyte maturation and functional integration, the feasibility of generating tissues for cardiac repair, and as models for pharmacology and toxicology bioassays. ECTs rapidly mature in vitro to acquire the features of functional cardiac muscle and respond to mechanical load with increased proliferation and maturation. ECTs are now being investigated as platforms for in vitro models for human diseases and for pharmacologic screening for drug toxicities. We tested the hypothesis that global ECT gene expression patterns are complex and sensitive to mechanical loading and tyrosine kinase inhibitors similar to the maturing myocardium. We generated ECTs from day 14.5 rat embryo ventricular cells, as previously published, and then conditioned constructs after 5 days in culture for 48 h with mechanical stretch (5%, 0.5 Hz) and/or the p38 MAPK (p38 mitogen-activated protein kinase) inhibitor BIRB796. RNA was isolated from individual ECTs and assayed using a standard Agilent rat 4 × 44k V3 microarray and Pathway Analysis software for transcript expression fold changes and changes in regulatory molecules and networks. Changes in expression were confirmed by quantitative-polymerase chain reaction (q-PCR) for selected regulatory molecules. At the threshold of a 1.5-fold change in expression, stretch altered 1559 transcripts, versus 1411 for BIRB796, and 1846 for stretch plus BIRB796. As anticipated, top pathways altered in response to these stimuli include cellular development, cellular growth and proliferation; tissue development; cell death, cell signaling, and small molecule biochemistry as well as numerous other pathways. Thus, ECTs display a broad spectrum of altered gene expression in response to mechanical load and/or tyrosine kinase inhibition, reflecting a complex regulation of proliferation, differentiation, and architectural alignment of cardiomyocytes and noncardiomyocytes within ECT.

Cardiac cell culture model as a left ventricle mimic for cardiac tissue generation.

A major challenge in cardiac tissue engineering is the delivery of hemodynamic mechanical cues that play a critical role in the early development and maturation of cardiomyocytes. Generation of functional cardiac tissue capable of replacing or augmenting cardiac function therefore requires physiologically relevant environments that can deliver complex mechanical cues for cardiomyocyte functional maturation. The goal of this work is the development and validation of a cardiac cell culture model (CCCM) microenvironment that accurately mimics pressure-volume changes seen in the left ventricle and to use this system to achieve cardiac cell maturation under conditions where mechanical loads such as pressure and stretch are gradually increased from the unloaded state to conditions seen in vivo. The CCCM platform, consisting of a cell culture chamber integrated within a flow loop was created to accomplish culture of 10 day chick embryonic ventricular cardiomyocytes subject to 4 days of stimulation (10 mmHg, ∼13% stretch at a frequency of 2 Hz). Results clearly show that CCCM conditioned cardiomyocytes accelerate cardiomyocyte structural and functional maturation in comparison to static unloaded controls as evidenced by increased proliferation, alignment of actin cytoskeleton, bundle-like sarcomeric α-actinin expression, higher pacing beat rate at lower threshold voltages, and increased shortening. These results confirm the CCCM microenvironment can accelerate immature cardiac cell structural and functional maturation for potential cardiac regenerative applications.

Gene transfer as a strategy to achieve permanent cardioprotection II: rAAV-mediated gene therapy with heme oxygenase-1 limits infarct size 1 year later without adverse functional consequences.

Extensive evidence indicates that heme oxygenase-1 (HO-1) exerts potent cytoprotective effects in response to stress. Previous studies have shown that gene therapy with HO-1 protects against myocardial ischemia/reperfusion injury for up to 8 weeks after gene transfer. However, the long-term effects of HO-1 gene therapy on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the HO-1 gene (rAAV/HO-1) that enables long-lasting transgene expression. Mice received injections in the anterior LV wall of rAAV/LacZ (LacZ group) or rAAV/HO-1 (HO-1 group); 1 year later, they were subjected to a 30-min coronary occlusion (O) and 4 h of reperfusion (R). Cardiac HO-1 gene expression was confirmed at 1 month and 1 year after gene transfer by immunoblotting and immunohistochemistry analyses. In the HO-1 group, infarct size (% of risk region) was dramatically reduced at 1 year after gene transfer (11.2 ± 2.1%, n = 12, vs. 44.7 ± 3.6%, n = 8, in the LacZ group; P < 0.05). The infarct-sparing effects of HO-1 gene therapy at 1 year were as powerful as those observed 24 h after ischemic PC (six 4-min O/4-min R cycles) (15.0 ± 1.7%, n = 10). There were no appreciable changes in LV fractional shortening, LV ejection fraction, or LV end-diastolic or end-systolic diameter at 1 year after HO-1 gene transfer as compared to the age-matched controls or with the LacZ group. Histology showed no inflammation in the myocardium 1 year after rAAV/HO-1-mediated gene transfer. These results demonstrate, for the first time, that rAAV-mediated HO-1 gene transfer confers long-term (1 year), possibly permanent, cardioprotection without adverse functional consequences, providing proof of principle for the concept of achieving prophylactic cardioprotection (i.e., "immunization against infarction").

Gene transfer as a strategy to achieve permanent cardioprotection I: rAAV-mediated gene therapy with inducible nitric oxide synthase limits infarct size 1 year later without adverse functional consequences.

The ultimate goal of prophylactic gene therapy is to confer permanent protection against ischemia. Although gene therapy with inducible nitric oxide synthase (iNOS) is known to protect against myocardial infarction at 3 days and up to 2 months, the long-term effects on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the iNOS gene (rAAV/iNOS), which enables long-lasting transgene expression. The ability of rAAV/iNOS to direct the expression of functional iNOS protein was confirmed in COS-7 cells before in vivo gene transfer. Mice received injections in the anterior LV wall of rAAV/LacZ or rAAV/iNOS; 1 year later, they underwent a 30-min coronary occlusion (O) and 4 h of reperfusion (R). iNOS gene transfer resulted in elevated iNOS protein expression (+3-fold vs. the LacZ group, n = 6; P < 0.05) and iNOS activity (+4.4-fold vs. the LacZ group, n = 6; P < 0.05) 1 year later. Infarct size (% of risk region) was dramatically reduced at 1 year after iNOS gene transfer (13.5 ± 2.2%, n = 12, vs. 41.7 ± 2.9%, n = 10, in the LacZ group; P < 0.05). The infarct-sparing effect of iNOS gene therapy at 1 year was as powerful as that observed 24 h after ischemic preconditioning (six 4-min O/4-min R cycles) (19.3 ± 2.3%, n = 11; P < 0.05). Importantly, compared with the LacZ group (n = 11), iNOS gene transfer (n = 10) had no effect on LV dimensions or function for up to 1 year (at 1 year: FS 34.5 ± 2.0 vs. 34.6 ± 2.6%, EF 57.0 ± 2.0 vs. 59.7 ± 2.9%, LVEDD 4.3 ± 0.1 vs. 4.2 ± 0.2 mm, LVESD 2.8 ± 0.1 vs. 2.9 ± 0.2 mm) (echocardiography). These data demonstrate, for the first time, that rAAV-mediated iNOS gene transfer affords long-term, probably permanent (1 year), cardioprotection without adverse functional consequences, providing a strong rationale for further preclinical testing of prophylactic gene therapy.

Intracoronary administration of cardiac stem cells in mice: a new, improved technique for cell therapy in murine models.

A model of intracoronary stem cell delivery that enables transgenesis/gene targeting would be a powerful tool but is still lacking. To address this gap, we compared intracoronary and intramyocardial delivery of lin(-)/c-kit(+)/GFP(+) cardiac stem cells (CSCs) in a murine model of reperfused myocardial infarction (MI). Lin(-)/c-kit(+)/GFP(+) CSCs were successfully expanded from GFP transgenic hearts and cultured with no detectable phenotypic change for up to ten passages. Intracoronary delivery of CSCs 2 days post-MI resulted in significant alleviation of adverse LV remodeling and dysfunction, which was at least equivalent, if not superior, to that achieved with intramyocardial delivery. Compared with intramyocardial injection, intracoronary infusion was associated with a more homogeneous distribution of CSCs in the infarcted region and a greater increase in viable tissue in this region, suggesting greater formation of new cardiomyocytes. Intracoronary CSC delivery resulted in improved function in the infarcted region, as well as in improved global LV systolic and diastolic function, and in decreased LV dilation and LV expansion index; the magnitude of these effects was similar to that observed after intramyocardial injection. We conclude that, in the murine model of reperfused MI, intracoronary CSC infusion is at least as effective as intramyocardial injection in limiting LV remodeling and improving both regional and global LV function. The intracoronary route appears to be superior in terms of uniformity of cell distribution, myocyte regeneration, and amount of viable tissue in the risk region. To our knowledge, this is the first study to report that intracoronary infusion of stem cells in mice is feasible and effective.

Chronic AMD3100 antagonism of SDF-1alpha-CXCR4 exacerbates cardiac dysfunction and remodeling after myocardial infarction.

The role of the SDF-1alpha-CXCR4 axis in response to myocardial infarction is unknown. We addressed it using the CXCR4 antagonist, AMD3100, to block SDF-1alpha interaction with CXCR4 after chronic coronary artery ligation. Chronic AMD3100 treatment decreased ejection fraction and fractional shortening in mice 20days after myocardial infarction compared with vehicle-treated mice (echocardiography). Morphometric analysis showed hearts of AMD3100-treated infarcted mice to have expanded scar, to be hypertrophic (confirmed by myocyte cross-section area) and dilated, with increased LV end systolic and end diastolic dimensions, and to have decreased scar collagen content; p-AKT levels were attenuated and this was accompanied by increased apoptosis. Despite increased injury, c-kit(pos) cardiac progenitor cells (CPCs) were increased in the risk region of AMD3100-treated infarcted mice; CPCs were CD34(neg)/CD45(neg) with the majority undergoing symmetric cell division. c-kit(pos)/MHC(pos) CPCs also increased in the risk region of the AMD3100-treated infarcted group. In this group, GSK-3beta signaling was attenuated compared to vehicle-treated, possibly accounting for increased proliferation and increased cardiac committed MHC(pos) CPCs. Increased proliferation following AMD3100 treatment was supported by increased levels of cyclin D1, a consequence of increased prolyl isomerase, Pin1, and decreased cyclin D1 phosphorylation. In summary, pharmacologic antagonism of CXCR4 demonstrates that SDF-1alpha-CXCR4 signaling plays an important role during and after myocardial infarction and that it exerts pleiotropic salubrious effects, protecting the myocardium from apoptotic cell death, facilitating scar formation, restricting CPC proliferation, and directing CPCs toward a cardiac fate.

Genetic ablation of luteinizing hormone receptor improves the amyloid pathology in a mouse model of Alzheimer disease.

Amyloid-beta peptide (Abeta) plays an essential pathophysiologic role in Alzheimer disease, and elevation of luteinizing hormone (LH) levels during aging has been implicated in its pathogenesis. To assess the effect of LH receptor deficiency on Abeta accumulation, we generated a bigenic mouse model, APPsw(+)/Lhr(-/-), which expresses human amyloid precursor protein (APPsw) in the background of LH receptor (Lhr) knockout. Genetic ablation of Lhr resulted in a significant decrease in the number of Abeta plaques and protein content in the hippocampus and cerebral cortex in both male and female mice. Accordingly, several Abeta deposition-related neuropathologic features and functionally relevant molecules were markedly improved, including decreased astrogliosis, reductions of elevated phosphorylated tau, c-fos, alpha7-nicotinic acetylcholine receptor, and restoration of the altered neuropeptide Y receptors Y1 and Y2. Diminution of Abeta accumulation in the absence of LH receptor supports the contention that dysregulation of LH may impact the pathogenesis of Alzheimer disease. The APPsw(+)/Lhr(-/-) mouse may be a useful tool for advancing understanding of the role of LH-mediated events in Alzheimer disease and a model in which to test therapeutic interventions.

Intracoronary administration of cardiac progenitor cells alleviates left ventricular dysfunction in rats with a 30-day-old infarction.

BACKGROUND: Administration of cardiac progenitor cells (CPCs) 4 hours after reperfusion ameliorates left ventricular function in rats with acute myocardial infarction (MI). Clinically, however, this approach is not feasible, because expansion of autologous CPCs after acute MI requires several weeks. Therefore, we sought to determine whether CPCs are beneficial in the more clinically relevant setting of an old MI (scar). METHODS AND RESULTS: One month after coronary occlusion/reperfusion, rats received an intracoronary infusion of vehicle or enhanced green fluorescent protein-labeled CPCs. Thirty-five days later, CPC-treated rats exhibited more viable myocardium in the risk region, less fibrosis in the noninfarcted region, and improved left ventricular function. Cells that stained positive for enhanced green fluorescent protein that expressed cardiomyocyte, endothelial, and vascular smooth muscle cell markers were observed only in 7 of 17 treated rats and occupied only 2.6% and 1.1% of the risk and noninfarcted regions, respectively. Transplantation of CPCs was associated with increased proliferation and expression of cardiac proteins by endogenous CPCs. CONCLUSIONS: Intracoronary administration of CPCs in the setting of an old MI produces beneficial structural and functional effects. Although exogenous CPCs can differentiate into new cardiac cells, this mechanism is not sufficient to explain the benefits, which suggests paracrine effects; among these, the present data identify activation of endogenous CPCs. This is the first report that CPCs are beneficial in the setting of an old MI when given by intracoronary infusion, the most widely applicable therapeutic approach in patients. Furthermore, this is the first evidence that exogenous CPC administration activates endogenous CPCs. These results open the door to new therapeutic applications for the use of autologous CPCs in patients with old MI and chronic ischemic cardiomyopathy.

Luteinizing hormone receptor deficiency increases the susceptibility to alkylating agent-induced lymphomagenesis in mice.

Previous studies have revealed a close link between luteinizing hormone (LH)/human chorionic gonadotropin (hCG) signaling and oncogenesis in gonadal and nongonadal tissues. To investigate whether genetic ablation of LH receptor (Lhr) affects the animal's oncogenic susceptibility, adult female wild-type (wt), heterozygous, and homozygous Lhr knockout (LhrKO) mice were intraperitoneally injected with an alkylating agent, N-methyl-N-nitrosourea (MNU, 50 mg/kg of body weight). The mice were sacrificed when they were short of breath or 10 months after the injection. The results showed that MNU induced non-Hodgkin's thymic and lymphonodus lymphomas in 70.6% and 100% of heterozygous and homozygous animals, respectively, compared with 35.7% in wt siblings. The tumor development was rapid; they were more aggressive and metastasized to the spleen, liver, and kidney in Lhr-deficient mice compared to wt siblings. All tumors were immunostained-positive for a T-cell specific marker, CD3, but not for a B-cell marker, CD22, suggesting that all the lymphomas arose from T-cells, which are known to be LH/hCG receptor-positive. There was no rearrangement of the Lhr gene locus or differences in thymic cell proliferation among the genotypes. However, apoptosis was lower in the Lhr-deficient thymuses. The thymic Bcl-2 levels were elevated and caspase-3 activation was reduced in Lhr heterozygous and homozygous animals. In conclusion, MNU induced a higher incidence and an earlier onset of aggressive lymphomas in LhrKO animals, which may be associated with a reduction in apoptosis of thymocytes.