Eliana C. Martinez

Epidermal Growth Factor Receptor Blockade Mediates Smooth Muscle Cell Apoptosis and Improves Survival in Rats With Pulmonary Hypertension

Background—We previously reported that administration of elastase inhibitors reverses fatal pulmonary arterial hypertension (PAH) in rats by inducing smooth muscle cell (SMC) apoptosis. We showed in pulmonary artery (PA) organ culture that the mechanism by which elastase inhibitors induce SMC apoptosis involves repression of matrix metalloproteinase (MMP) activity and subsequent signaling through &agr;v&bgr;3-integrins and epidermal growth factor receptors (EGFRs). This suggests that blockade of these downstream effectors may also induce regression of PAH. Methods and Results—In this study, we first showed in PA organ culture that MMP inhibition or &agr;v&bgr;3-integrin blockade with agents in clinical and preclinical use (SC-080 and cilengitide, respectively) mediates SMC apoptosis and regression of medial hypertrophy. We also documented similar results with an EGFR tyrosine kinase inhibitor. We then induced PAH in rats by injection of monocrotaline and, at day 21, began a 2-week treatment with SC-080, cilengitide, or the EGFR inhibitor PKI166. No vehicle- or cilengitide-treated animal survived beyond 2 weeks. Administration of SC-080 resulted in 44% survival at 2 weeks, and PKI166 therapy resulted in 78% and 54% survival in daily or 3-times-weekly treated animals, respectively. Four weeks after cessation of PKI166, we documented survivals of 50% and 23% in the 2 treatment groups, associated with reductions in pulmonary pressure, right ventricular hypertrophy, and abnormally muscularized distal arteries. Conclusion—We propose that selective blockade of EGFR signaling may be a novel strategy to reverse progressive, fatal PAH.

Novel Injectable Bioartificial Tissue Facilitates Targeted, Less Invasive, Large-Scale Tissue Restoration on the Beating Heart After Myocardial Injury

Background—Implantation of bioartificial patches distorts myocardial geometry, and functional improvement of the recipient heart is usually attributed to reactive angiogenesis around the graft. With the liquid bioartificial tissue compound used in this study, we achieved targeted large-scale support of the infarcted left ventricular wall and improvement of heart function. Methods and Results—A liquid compound consisting of growth factor-free Matrigel and 106 green fluorescent protein (GFP)-positive mouse (129sv) embryonic stem cells (ESCs) was generated and injected into the area of ischemia after ligation of the left anterior descending artery in BALB/c mice (group I). Left anterior descending artery-ligated mice (group II) and mice with Matrigel (group III) or ESC treatment alone (group IV) were used as the control groups (n=5 in all groups). The hearts were harvested for histology 2 weeks later after echocardiographic assessment with a 15-MHz probe. The liquid injectable tissue solidified at body temperature and retained the geometry of the infarcted lateral wall. Immunofluorescence stains revealed voluminous GFP grafts. The quality of restoration (graft/infarct area ratio) was 45.5±10.8% in group I and 29.1±6.7% in group IV (P=0.034). ESCs expressed connexin 43 at intercellular contact sites. The mice treated with the compound had a superior heart function compared with the controls (P<0.0001 by ANOVA/Bonferroni test; group I: 27.1±5.4, group II:11.9±2.4, group III:16.2±2.8, group IV: 19.1±2.7). Conclusions—Injectable bioartificial tissue restores the heart’s geometry and function in a targeted and nondistorting fashion. This new method paves the way for novel interventional approaches to myocardial repair, using both stem cells and matrices.

Interdependent serotonin transporter and receptor pathways regulate S100A4/Mts1, a gene associated with pulmonary vascular disease

Heightened expression of the S100 calcium–binding protein, S100A4/Mts1, is observed in pulmonary vascular disease. Loss of serotonin (5-hydroxytryptamine [5-HT]) receptors or of the serotonin transporter (SERT) attenuates pulmonary hypertension in animals, and polymorphisms causing gain of SERT function are linked to clinical pulmonary vascular disease. Because 5-HT induces release of S100&bgr;, we investigated the codependence of 5-HT receptors and SERT in regulating S100A4/Mts1 in human pulmonary artery smooth muscle cells (hPA-SMC). 5-HT elevated S100A4/Mts1 mRNA levels and increased S100A4/Mts1 protein in hPA-SMC lysates and culture media. S100A4/Mts1 in the culture media stimulated proliferation and migration of hPA-SMC in a manner dependent on the receptor for advanced glycation end products. Treatment with SB224289 (selective antagonist of 5-HT1B), fluoxetine (SERT inhibitor), SERT RNA-interference, and iproniazid (monoamine oxidase-A inhibitor), blocked 5-HT–induced S100A4/Mts1. 5-HT signaling mediated phosphorylation (p) of extracellular signal–regulated kinase 1/2 (pERK1/2), but pERK1/2 nuclear translocation depended on SERT, monoamine oxidase activity, and reactive oxygen species. Nuclear translocation of pERK1/2 was required for pGATA-4–mediated transcription of S100A4/Mts1. These data provide evidence for a mechanistic link between the 5-HT pathway and S100A4/Mts1 in pulmonary hypertension and explain how the 5-HT1B receptor and SERT are codependent in regulating S100A4/Mts1.

Myocardial tissue engineering: the quest for the ideal myocardial substitute

There has been an intense and competitive quest to manufacture bioartificial heart muscle in the last decade. Numerous biocompatible scaffolds and scaffold-free systems, enriched with various cell types, have been used to fabricate 3D grafts for myocardial repair. In spite of the impressive achievements in the myocardial tissue-engineering field, many issues remain to be addressed before clinical application of this strategy becomes feasible. This is largely due to the uniqueness of the heart’s structure and function. This review provides a survey upon the reported strategies, and indicates caveats and perspectives in the field of myocardial tissue engineering.

Off-pump coronary bypass grafting is safe and efficient in patients with left main disease and higher EuroScore.

BACKGROUND Summary Left main disease (LMD) and associated cardiac risk factors are often perceived as a limiting factor for the outcome of off-pump coronary artery bypass (OPCAB) grafting. In this study, we assess whether the outcome of OPCAB surgery is affected in such patients. METHODS We retrospectively compared perioperative parameters in 66 OPCAB patients (group A) with LMD and 216 OPCAB patients without (group B) LMD. The patients were operated in the time frame between 2002 and 2007. LMD was defined as a stenosis >50%. RESULTS Patients in group A had a higher EuroSCORE (logistic: 3.7+/-0.1 vs 6.3+/-0.3, p=0.027), increased coronary artery disease (CAD) family history (p=0.015) and cerebrovascular accidents (p=0.027), increased history of congestive heart failure (p=0.013), more urgent surgery (p=0.008), previous percutaneous transluminal coronary angioplasties (PTCAs) (p=0.05) and previous stent implantation (p=0.023). An intra-aortic balloon pump (IABP) was inserted more frequently in the LMD group preoperatively (p=0.004). There were two conversions to on-pump during OPCAB surgery. There were no differences in the postoperative outcomes in the LMD group A versus group B, such as cardiac-related events, neurological deficits, cardiac enzyme course, arrhythmias, blood loss, infections and renal failure. CONCLUSIONS The presence of LMD and higher EuroSCORE does not yield adverse outcomes in OPCAB patients.

GYY4137 attenuates remodeling, preserves cardiac function and modulates the natriuretic peptide response to ischemia

AIMS Myocardial infarction followed by adverse left ventricular (LV) remodeling is the most frequent proximate cause of heart failure. Hydrogen sulfide (H2S) is an important endogenous modulator of diverse physiological and pathophysiological processes. Its role in post-ischemic ventricular remodeling and the associated neurohormonal responses has not been defined. Here, we aimed at evaluating whether the slow-releasing water-soluble H2S donor GYY4137 (GYY) exerts cardioprotective effects and modulates the neurohormonal response to cardiac ischemic injury. METHODS AND RESULTS Treatment for 2 or 7 days with GYY (100 mg/Kg/48 h, IP) after acute myocardial infarction (MI) in rats preserved LV dimensions and function in vivo, compared to untreated infarcted (MI), placebo- and dl-propargylglycine- (PAG, an inhibitor of endogenous H2S synthesis) treated animals (n=9/group/time-point). LV dimensions and function in GYY-treated animals were comparable to healthy sham-operated rats. GYY-treated hearts had significantly less LV fibrosis than MI, placebo and PAG hearts. A higher density of blood vessels was found in the LV scar area of GYY-treated animals compared to all other infarcted groups. Despite preserved LV structure and function, treatment with GYY increased the levels of the natriuretic peptides ANP and BNP in association with enhanced cyclic GMP levels, paralleled by higher cGMP-dependent protein kinase type I (cGKI) protein levels. CONCLUSIONS Our data suggest that the slow-releasing H2S donor, GYY4137, preserves cardiac function, attenuates adverse remodeling and may exert post-ischemic cardioprotective (pro-angiogenic, anti-apoptotic, anti-hypertrophic and anti-fibrotic) effects in part through enhanced early post-ischemic endogenous natriuretic peptide activation.

Cord Lining-Mesenchymal Stem Cells Graft Supplemented with an Omental Flap Induces Myocardial Revascularization and Ameliorates Cardiac Dysfunction in a Rat Model of Chronic Ischemic Heart Failure

Myocardial restoration using tissue-engineered grafts to regenerate the ischemic myocardium offers improved donor cell retention, yet a limited cell survival resulting from poor vascularization needs to be addressed. A cell type derived from the subamnion, namely, cord-lining mesenchymal stem cells (CL-MSC), has recently been identified. Here we present a restorative strategy that combines a fibrin graft containing human CL-MSC and omental flap providing, thereby, cell-, structural-, and angiogenic support to the injured myocardium. The graft consisted of a mixture of 2×10(6) CL-MSC-GFP-Fluc and fibrin. Myocardial infarction (MI) was induced in nude rats and following confirmation of ensued heart failure with echocardiography 2 weeks after injury, therapeutic intervention was performed as follows: untreated (MI, n=7), CL-MSC graft (CL-MSCG, n=8), CL-MSCG and omental flap (CL-MSCG+OM, n=11), and omental flap (OM, n=8). In vivo bioluminescence imaging at 1, 3, 7, and 14 days post-treatment indicated comparable early donor cell viability between the CL-MSCG and CL-MSCG+OM. Treatment with CL-MSCG+OM improved the myocardial function as assessed by the measurement of end-diastolic left ventricular (LV) pressure (3.53±0.34 vs. 5.21±0.54 mmHg, p<0.05), contractility (+dP/dt, 3383.8±250.78 mmHg vs. 2464.9±191.8 mmHg, p<0.05), and the relaxation rate (-dP/dt, -2707.2±250.7 mmHg vs. 1948.7±207.8 mmHg, p<0.05), compared to MI control 6 weeks after ischemic injury. Furthermore, evidence of a 20.32% increase in the ejection fraction was observed in CL-MSCG+OM rats from week 2 to 6 after injury. Both CL-MSCG and CL-MSCG+OM led to an enhanced cardiac output (p<0.05), and attenuated the infarct size (35.7%±4.2% and 34.7%±4.8%), as compared to MI (60.7%±3.1%; p<0.01 and p<0.001, respectively). All treated groups had a higher arteriole density than controls. Yet, a higher amount of functional blood vessels, and a 20-fold increase in arteriole numbers were found in CL-MSCG+OM. Altogether, CL-MSCGs supplemented with vascular supply have the potential to repair the failing, chronically ischemic heart by improving myocardial revascularization, attenuating remodeling, and ameliorating cardiac dysfunction.

Grafts Enriched with Subamnion-Cord-Lining Mesenchymal Stem Cell Angiogenic Spheroids Induce Post-Ischemic Myocardial Revascularization and Preserve Cardiac Function in Failing Rat Hearts

A crucial question in post-ischemic cell therapy refers to the ideal method of cell delivery to the heart. We hypothesized that epicardial implantation of subamnion-cord-lining mesenchymal stem cells (CL-MSC) angiogenic spheroids embedded within fibrin grafts (SASG) facilitates donor cell survival and enhances cardiac function in failing rat hearts. Furthermore, we compared the efficacy of this approach applied through two delivery methods. Spheroids made of 1.5×10(4) human CL-MSC coated with 2×10(3) human umbilical vein endothelial cells were self-assembled in hanging drops. SASG were constructed by embedding 150 spheroids in fibrin matrix. Except for untreated rats (MI, n=8), grafts were implanted 2 weeks after myocardial infarction upon confirmation of ensued heart failure through thoracotomy: SASG (n=8) and fibrin graft (FG, n=8); or video-assisted thoracoscopic surgery (VATS): SASG-VATS (n=8) and FG-VATS (n=7). In vivo CL-MSC survival was comparable between both SASG-treated groups throughout the study. SASG and SASG-VATS animals had decreased left ventricular end-diastolic pressure relative to untreated animals, and increased fractional shortening compared to MI and FG controls, 4 weeks after treatment. A 14.1% and 6.2% enhancement in ejection fraction from week 2 to 6 after injury was observed in SASG/SASG-VATS, paralleled by improvement in cardiac output. Treated hearts had smaller scar size, and more blood vessels than MI, while donor CL-MSC contributed to arteriogenesis within the graft and infarct areas. Taken together, our data suggest that SASG treatment has the potential to restore failing hearts by preserving cardiac function and inducing myocardial revascularization, while attenuating cardiac fibrosis. Furthermore, we introduce a method for minimally invasive in situ graft assembly.

Natriuretic peptide receptor 3 (NPR3) is regulated by microRNA-100

Natriuretic peptide receptor 3 (NPR3) is the clearance receptor for the cardiac natriuretic peptides (NPs). By modulating the level of NPs, NPR3 plays an important role in cardiovascular homeostasis. Although the physiological functions of NPR3 have been explored, little is known about its regulation in health or disease. MicroRNAs play an essential role in the post-transcriptional expression of many genes. Our aim was to investigate potential microRNA-based regulation of NPR3 in multiple models. Hypoxic challenge elevated levels of NPPB and ADM mRNA, as well as NT-proBNP and MR-proADM in human left ventricle derived cardiac cells (HCMa), and in the corresponding conditioned medium, as revealed by qRT-PCR and ELISA. NPR3 was decreased while NPR1 was increased by hypoxia at mRNA and protein levels in HCMa. Down-regulation of NPR3 mRNA was also observed in infarct and peri-infarct cardiac tissue from rats undergoing myocardial infarction. From microRNA microarray analyses and microRNA target predictive databases, miR-100 was selected as a candidate regulator of NPR3 expression. Further analyses confirmed up-regulation of miR-100 in hypoxic cells and associated conditioned media. Antagomir-based silencing of miR-100 enhanced NPR3 expression in HCMa. Furthermore, miR-100 levels were markedly up-regulated in rat hearts and in peripheral blood after myocardial infarction and in the blood from heart failure patients. Results from this study point to a role for miR-100 in the regulation of NPR3 expression, and suggest a possible therapeutic target for modulation of NP bioactivity in heart disease.

An autologous platelet-rich plasma hydrogel compound restores left ventricular structure, function and ameliorates adverse remodeling in a minimally invasive large animal myocardial restoration model: A translational approach: Vu and Pal “Myocardial Repair: PRP, Hydrogel and Supplements”

AIMS Cell-based myocardial restoration has not penetrated broad clinical practice yet due to poor cell retention and survival rates. In this study, we attempt a translational, large-scale restorative but minimally invasive approach in the pig, aiming at both structurally stabilizing the left ventricular (LV) wall and enhancing function following ischemic injury. METHODS AND RESULTS A myocardial infarction (MI) was created by permanent ligation of left circumflex coronary artery through a small lateral thoracotomy. Thirty-six Yorkshire pigs were randomized to receive transthoracic intramyocardial injection into both infarct and border zone areas with different compounds: 1) Hyaluronic acid-based hydrogel; 2) autologous platelet-rich plasma (PRP); 3) ascorbic acid-enriched hydrogel (50 mg/L), combined with IV ibuprofen (25 mg/kg) and allopurinol (25 mg/kg) (cocktail group); 4) PRP and cocktail (full-compound); or 5) saline (control). The latter two groups received daily oral ibuprofen (25 mg/kg) for 7 days and allopurinol (25 mg/kg) for 30 days, postoperatively. Hemodynamic and echocardiographic studies were carried out at baseline, immediately after infarction and at end-point. Eight weeks after MI, the full-compound group had better LV fractional area change, ejection fraction and smaller LV dimensions than the control group. Also, dp/dtmax was significantly higher in the full-compound group when the heart rate increased from 100 bpm to 160bpm in stress tests. Blood vessel density was higher in the full-compound group, compared to the other treatment groups. CONCLUSIONS A combination of PRP, anti-oxidant and anti-inflammatory factors with intramyocardial injection of hydrogel has the potential to structurally and functionally improve the injured heart muscle while attenuating adverse cardiac remodeling after acute myocardial infarction.