UNVEILING NOVEL CARDIOPROTECTIVE EFFECTS OF GYY4137, A SLOW-RELEASING HYDROGEN SULFIDE DONOR, AND THEIR RELATIONSHIP TO NEUROHORMONAL MODULATION AND MICRORNA PROFILES DURING MYOCARDIAL ISCHEMIC INJURY.

Master'sMASTER OF SCIENC

Post‐ischaemic angiogenic therapy using in vivo prevascularized ascorbic acid‐enriched myocardial artificial grafts improves heart function in a rat model

Angiogenesis plays a key role in post‐ischaemic myocardial repair. We hypothesized that epicardial implantation of an ascorbic acid (AA)‐enriched myocardial artificial graft (MAG), which has been prevascularized in the recipients' own body, promotes restoration of the ischaemic heart. Gelatin patches were seeded with GFP–luciferase‐expressing rat cardiomyoblasts and enriched with 5 μm AA. Grafts were prevascularized in vivo for 3 days, using a renal pouch model in rats. The MAG patch was then implanted into the same rat's ischaemic heart following myocardial infarction (MI). MAG‐treated animals (MAG group, n = 6) were compared to untreated infarcted animals as injury controls (MI group, n = 6) and sham‐operated rats as healthy controls (healthy group, n = 7). In vivo bioluminescence imaging indicated a decrease in donor cell survival by 83% during the first week post‐implantation. Echocardiographic and haemodynamic assessment 4 weeks after MI revealed that MAG treatment attenuated left ventricular (LV) remodelling (LV end‐systolic volume, 0.31 ± 0.13 vs 0.81 ± 0.01 ml, p < 0.05; LV end‐diastolic volume 0.79 ± 0.33 vs 1.83 ± 0.26 ml, p < 0.076) and preserved LV wall thickness (0.21 ± 0.03 vs 0.09 ± 0.005 cm, p < 0.05) compared to the MI group. Cardiac output was higher in MAG than MI (51.59 ± 6.5 vs 25.06 ± 4.24 ml/min, p < 0.01) and comparable to healthy rats (47.08 ± 1.9 ml/min). Histology showed decreased fibrosis, and a seven‐fold increase in blood vessel density in the scar area of MAG compared to MI group (15.3 ± 1.1 vs 2.1 ± 0.3 blood vessels/hpf, p < 0.0001). Implantation of AA‐enriched prevascularized grafts enhanced vascularity in ischaemic rat hearts, attenuated LV remodelling and preserved LV function. Copyright © 2011 John Wiley & Sons, Ltd

MicroRNA-31 promotes adverse cardiac remodeling and dysfunction in ischemic heart disease

Myocardial infarction (MI) triggers a dynamic microRNA response with the potential of yielding therapeutic targets. We aimed to identify novel aberrantly expressed cardiac microRNAs post-MI with potential roles in adverse remodeling in a rat model, and to provide post-ischemic therapeutic inhibition of a candidate pathological microRNA in vivo. Following microRNA array profiling in rat hearts 2 and 14days post-MI, we identified a time-dependent up-regulation of miR-31 compared to sham-operated rats. A progressive increase of miR-31 (up to 91.4±11.3 fold) was detected in the infarcted myocardium by quantitative real-time PCR. Following target prediction analysis, reporter gene assays confirmed that miR-31 targets the 3´UTR of cardiac troponin-T (Tnnt2), E2F transcription factor 6 (E2f6), mineralocorticoid receptor (Nr3c2) and metalloproteinase inhibitor 4 (Timp4) mRNAs. In vitro, hypoxia and oxidative stress up-regulated miR-31 and suppressed target genes in cardiac cell cultures, whereas LNA-based oligonucleotide inhibition of miR-31 (miR-31i) reversed its repressive effect on target mRNAs. Therapeutic post-ischemic administration of miR-31i in rats silenced cardiac miR-31 and enhanced expression of target genes, while preserving cardiac structure and function at 2 and 4weeks post-MI. Left ventricular ejection fraction (EF) improved by 10% (from day 2 to 30 post-MI) in miR-31i-treated rats, whereas controls receiving scrambled LNA inhibitor or placebo incurred a 17% deterioration in EF. miR-31i decreased end-diastolic pressure and infarct size; attenuated interstitial fibrosis in the remote myocardium and enhanced cardiac output. miR-31 induction after MI is deleterious to cardiac function while its therapeutic inhibition in vivo ameliorates cardiac dysfunction and prevents the development of post-ischemic adverse remodeling

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

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. Treatment for 2 or 7days with GYY (100mg/Kg/48h, 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. 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. •Here, we assessed the role of H2S in modulating neurohormonal responses to ischemic injury.•We used the slow-releasing H2S donor, GYY4137 (GYY), to treat rats after myocardial infarction.•Treatment with GYY preserves cardiac function, reduces LV fibrosis and promotes angiogenesis.•GYY may exert cardioprotective actions through 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

Variability in Microplate Surface Properties and Its Impact on ELISA

10.1373/jalm.2017.023952The Journal of Applied Laboratory Medicine: An AACC Publication25687-69

Epitope-directed monoclonal antibody production using a mixed antigen cocktail facilitates antibody characterization and validation

10.1038/s42003-021-01965-xCOMMUNICATIONS BIOLOGY4

Epitope-directed monoclonal antibody production using a mixed antigen cocktail facilitates antibody characterization and validation (vol 6, 441, 2021)

10.1038/s42003-021-02091-4COMMUNICATIONS BIOLOGY4