Deborah Vela

Mesenchymal Stem Cells Differentiate into an Endothelial Phenotype, Enhance Vascular Density, and Improve Heart Function in a Canine Chronic Ischemia Model

Background—Bone marrow–derived stem cells are under investigation as a treatment for ischemic heart disease. Mesenchymal stem cells (MSCs) have been used preferentially in the acute ischemia model; data in the chronic ischemia model are lacking. Methods and Results—Twelve dogs underwent ameroid constrictor placement. Thirty days later, they received intramyocardial injections of either MSCs (100×106 MSCs/10 mL saline) (n=6) or saline only (10 mL) (controls) (n=6). All were euthanized at 60 days. Resting and stress 2D echocardiography was performed at 30 and 60 days after ameroid placement. White blood cell count (WBC), C-reactive protein (CRP), creatine kinase MB (CK-MB), and troponin I levels were measured. Histopathological and immunohistochemical analyses were performed. Mean left ventricular ejection fraction was similar in both groups at baseline but significantly higher in treated dogs at 60 days. WBC and CRP levels were similar over time in both groups. CK-MB and troponin I increased from baseline to 48 hours, eventually returning to baseline. There was a trend toward reduced fibrosis and greater vascular density in the treated group. MSCs colocalized with endothelial and smooth muscle cells but not with myocytes. Conclusions—In a canine chronic ischemia model, MSCs differentiated into smooth muscle cells and endothelial cells, resulting in increased vascularity and improved cardiac function.

Return to the fetal gene program

A hallmark of cardiac metabolism before birth is the predominance of carbohydrate use for energy provision. After birth, energy substrate metabolism rapidly switches to the oxidation of fatty acids. This switch accompanies the expression of “adult” isoforms of metabolic enzymes and other proteins. However, in a variety of pathophysiologic conditions, including hypoxia, ischemia, hypertrophy, atrophy, diabetes, and hypothyroidism, the postnatal heart returns to the “fetal” gene program. These adaptive mechanisms are also a feature of the failing heart muscle, where at a certain point this fetal‐like reprogramming no longer suffices to support cardiac structure and function. We advance the hypothesis that in the postnatal heart, metabolic remodeling triggers the process through glycosylation of transcription factors, potentially protecting the stressed heart from irreversible functional impairment and programmed cell death. In other words, we propose a metabolic link to gene expression in the heart.

Cardiomyocyte death and renewal in the normal and diseased heart

During post-natal maturation of the mammalian heart, proliferation of cardiomyocytes essentially ceases as cardiomyocytes withdraw from the cell cycle and develop blocks at the G0/G1 and G2/M transition phases of the cell cycle. As a result, the response of the myocardium to acute stress is limited to various forms of cardiomyocyte injury, which can be modified by preconditioning and reperfusion, whereas the response to chronic stress is dominated by cardiomyocyte hypertrophy and myocardial remodeling. Acute myocardial ischemia leads to injury and death of cardiomyocytes and nonmyocytic stromal cells by oncosis and apoptosis, and possibly by a hybrid form of cell death involving both pathways in the same ischemic cardiomyocytes. There is increasing evidence for a slow, ongoing turnover of cardiomyocytes in the normal heart involving death of cardiomyocytes and generation of new cardiomyocytes. This process appears to be accelerated and quantitatively increased as part of myocardial remodeling. Cardiomyocyte loss involves apoptosis, autophagy, and oncosis, which can occur simultaneously and involve different individual cardiomyocytes in the same heart undergoing remodeling. Mitotic figures in myocytic cells probably represent maturing progeny of stem cells in most cases. Mitosis of mature cardiomyocytes that have reentered the cell cycle appears to be a rare event. Thus, cardiomyocyte renewal likely is mediated primarily by endogenous cardiac stem cells and possibly by blood-born stem cells, but this biological phenomenon is limited in capacity. As a consequence, persistent stress leads to ongoing remodeling in which cardiomyocyte death exceeds cardiomyocyte renewal, resulting in progressive heart failure. Intense investigation currently is focused on cell-based therapies aimed at retarding cardiomyocyte death and promoting myocardial repair and possibly regeneration. Alteration of pathological remodeling holds promise for prevention and treatment of heart failure, which is currently a major cause of morbidity and mortality and a major public health problem. However, a deeper understanding of the fundamental biological processes is needed in order to make lasting advances in clinical therapeutics in the field.

Markers of Autophagy Are Downregulated in Failing Human Heart after Mechanical Unloading

Background— Autophagy is a molecular process that breaks down damaged cellular organelles and yields amino acids for de novo protein synthesis or energy provision. Mechanical unloading with a left ventricular assist device (LVAD) decreases the energy demand of the failing human heart. We tested the hypothesis that LVAD support reverses activation of autophagy. Methods and Results— Paired biopsy samples of left ventricular myocardium were obtained from 9 patients with idiopathic dilated cardiomyopathy (mean duration of LVAD support, 214 days) at the time of implantation and explantation of the LVAD. Transcript and protein levels of markers and mediators of autophagy and apoptosis were measured by quantitative reverse-transcription polymerase chain reaction and Western blotting. TUNEL assays, C9 immunohistochemistry, and 20S proteasome activity assays were also performed. Mechanical unloading significantly decreased mRNA transcript levels of Beclin-1, autophagy-related gene 5 (Atg5), and microtubule-associated protein-1 light chain-3 (MAP1-LC3 or LC3; P<0.02). Protein levels of Beclin-1, Atg5–Atg12 conjugate, and LC3-II were also significantly reduced after LVAD support (P<0.05). A significant increase in 20S proteasome activity was observed with unloading, in parallel to the decrease in autophagic markers. Although BNIP3 and the ratio of activated caspase 3 to procaspase 3 increased after LVAD support, Bcl-2 and TUNEL-positive nuclei were not significantly different between samples. Conclusions— Mechanical unloading of the failing human heart decreases markers of autophagy. These findings suggest that autophagy may be an adaptive mechanism in the failing heart, and this phenomenon is attenuated by LVAD support.

The role of periadventitial fat in atherosclerosis.

CONTEXT It has become increasingly evident that adipose tissue is a multifunctional organ that produces and secretes multiple paracrine and endocrine factors. Research into obesity, insulin resistance, and diabetes has identified a proinflammatory state associated with obesity. Substantial differences between subcutaneous and omental fat have been noted, including the fact that omental fat produces relatively more inflammatory cytokines. Periadventitial fat, as a specific adipose tissue subset, has been overlooked in the field of atherosclerosis despite its potential diagnostic and therapeutic implications. OBJECTIVE To review (1) evidence for the role of adventitial and periadventitial fat in vessel remodeling after injury, (2) the relationship between adventitial inflammation and atherosclerosis, (3) the association between periadventitial fat and plaque inflammation, and (4) the diagnostic and therapeutic implications of these roles and relationships for the progression of atherosclerosis. DATA SOURCES We present new data showing greater uptake of iron, administered in the form of superparamagnetic iron oxide, in the periadventitial fat of atherosclerotic mice than in control mice. In addition, macrophage density in the periadventitial fat of lipid-rich plaques is increased compared with fibrocalcific plaques. CONCLUSIONS There is a striking paucity of data on the relationship between the periadventitial fat of coronary arteries and atherosclerosis. Greater insight into this relationship might be instrumental in making strides into the pathophysiology, diagnosis, and treatment of coronary artery disease.

ULTRASOUND-ENHANCED rt-PA THROMBOLYSIS IN AN EX VIVO PORCINE CAROTID ARTERY MODEL

Ultrasound is known to enhance recombinant tissue plasminogen activator (rt-PA) thrombolysis. In this study, occlusive porcine whole blood clots were placed in flowing plasma within living porcine carotid arteries. Ultrasonically induced stable cavitation was investigated as an adjuvant to rt-PA thrombolysis. Aged, retracted clots were exposed to plasma alone, plasma containing rt-PA (7.1 ± 3.8 μg/mL) or plasma with rt-PA and Definity® ultrasound contrast agent (0.79 ± 0.47 μL/mL) with and without 120-kHz continuous wave ultrasound at a peak-to-peak pressure amplitude of 0.44 MPa. An insonation scheme was formulated to promote and maximize stable cavitation activity by incorporating ultrasound quiescent periods that allowed for the inflow of Definity®-rich plasma. Cavitation was measured with a passive acoustic detector throughout thrombolytic treatment. Thrombolytic efficacy was measured by comparing clot mass before and after treatment. Average mass loss for clots exposed to rt-PA and Definity® without ultrasound (n = 7) was 34%, and with ultrasound (n = 6) was 83%, which constituted a significant difference (p < 0.0001). Without Definity® there was no thrombolytic enhancement by ultrasound exposure alone at this pressure amplitude (n = 5, p < 0.0001). In the low-oxygen environment of the ischemic artery, significant loss of endothelium occurred but no correlation was observed between arterial tissue damage and treatment type. Acoustic stable cavitation nucleated by an infusion of Definity® enhances rt-PA thrombolysis without apparent treatment-related damage in this ex vivo porcine carotid artery model.

Ultrasound-enhanced delivery of targeted echogenic liposomes in a novel ex vivo mouse aorta model

The goal of this study was to determine whether targeted, Rhodamine-labeled echogenic liposomes (Rh-ELIP) containing nanobubbles could be delivered to the arterial wall, and whether 1-MHz continuous wave ultrasound would enhance this delivery profile. Aortae excised from apolipoprotein-E-deficient (n=8) and wild-type (n=8) mice were mounted in a pulsatile flow system through which Rh-ELIP were delivered in a stream of bovine serum albumin. Half the aortae from each group were treated with 1-MHz continuous wave ultrasound at 0.49 MPa peak-to-peak pressure, and half underwent sham exposure. Ultrasound parameters were chosen to promote stable cavitation and avoid inertial cavitation. A broadband hydrophone was used to monitor cavitation activity. After treatment, aortic sections were prepared for histology and analyzed by an individual blinded to treatment conditions. Delivery of Rh-ELIP to the vascular endothelium was observed, and sub-endothelial penetration of Rh-ELIP was present in five of five ultrasound-treated aortae and was absent in those not exposed to ultrasound. However, the degree of penetration in the ultrasound-exposed aortae was variable. There was no evidence of ultrasound-mediated tissue damage in any specimen. Ultrasound-enhanced delivery within the arterial wall was demonstrated in this novel model, which allows quantitative evaluation of therapeutic delivery.

Influenza virus directly infects, inflames, and resides in the arteries of atherosclerotic and normal mice

OBJECTIVE Influenza can trigger heart attacks, and vaccination against influenza reduces the risk of cardiovascular events. Currently, it is believed that influenza virus in general does not disseminate to extra-pulmonary tissues. We assessed the vascular effects of influenza infection and whether the virus can directly infect atherosclerotic arteries in mice. METHODS/RESULTS We intranasally infected 4 different types of mice--atherosclerotic apo E-deficient (our primary model), LDL receptor knockout, C57BL/6, and outbred Swiss--with influenza A/HK (H3/N2) virus. On day 7 after infection, we cultured viable virus from lung, aorta, and heart tissue, but not from the blood of apo E-deficient mice. Immunofluorescence studies showed influenza A virus NP1 protein and real time polymerase chain reaction (PCR) assay showed RNA in the aorta of infected apo E-deficient mice. Infected mice had significantly higher blood levels of chemokines and cytokines than control mice. At the local level, gene expression for several chemokines and cytokines was increased and eNOS expression was decreased. Infected mice had a higher density of macrophages in plaque than did control mice. CONCLUSIONS We have shown for the first time that influenza virus can directly infect and reside in atherosclerotic arteries and that infection was associated with systemic and arterial-level pro-inflammatory changes.

Immunologic and inflammatory reactions to exogenous stem cells: Implications for experimental studies and clinical trials for myocardial repair

Intense research is under way to determine the optimal stem cell type and regimen for repairing diseased myocardium. Although initial studies in humans focused on the use of homologous stem cells, allogeneic or xenogeneic stem cells have been studied extensively in experimental work. Clinical trials with allogeneic stem cells are now under way, an approach based on the premise that stem cells and precursor cells are characterized as being immunotolerant. However, evidence indicates that stem cells may gain immune potency in vivo, especially when delivered to inflamed tissue, such as acutely infarcted myocardium. Histopathologic studies show the presence of a lymphohistiocytic inflammatory reaction at the sites of delivery of allogeneic stem cells, a response that is exaggerated with the use of xenogeneic stem cells. The immune-mediated inflammatory reaction to allogeneic and xenogeneic stem cells may elicit a spectrum of effects, ranging from beneficial (e.g., increased paracrine activity) to detrimental (e.g., accelerated damage and removal of stem cells). Although the issue of immune-mediated inflammatory responses to non-self stem cells requires further evaluation, non-self stem cells should not be considered as immunologically inert or exclusively immunosuppressive in vivo.

Histopathological Study of Healing After Allogenic Mesenchymal Stem Cell Delivery in Myocardial Infarction in Dogs

In this histological study, we assessed the role of mesenchymal stem cells (MSCs) in the healing process that takes place during the subacute phase of myocardial infarction in dogs. Seven days after occlusion of the left anterior descending coronary artery, adult mongrel dogs received 100 × 106 4′-6-diamidino-2-phenylindole (DAPI)-labeled allogenic bone marrow-derived MSCs by the transendocardial (TE, n = 6) and intracoronary (IC, n = 4) routes; control dogs (n = 6) received no infusion. The dogs were euthanized at 21 days after occlusion. Hearts were excised and sliced from apex to base into four transverse sections, which were divided into nine segments. Paraffin sections from each segment were stained with hematoxylin and eosin, trichrome, picrosirius red, and antibodies against several extracellular matrix components. Frozen sections were immunostained for host cardiac phenotypical markers and analyzed by epifluorescence and deconvolution fluorescence microscopy (DFM). We found less unresolved necrotic myocardium and more extracellular matrix deposition in MSC-treated dogs than in controls 2 weeks after cell delivery. By DFM, no DAPI+ MSC nuclei were observed within native cardiac cells. MSCs delivered during the subacute phase of acute myocardial infarction positively affect healing, apparently by mechanisms other than differentiation into mature native cardiac cells.