BackgroundCardiomyocyte‐specific transgenic mice overexpressing S100A6, a member of the family of EF‐hand calcium‐binding proteins, develop less cardiac hypertrophy, interstitial fibrosis, and myocyte apoptosis after permanent coronary ligation, findings that support S100A6 as a potential therapeutic target after acute myocardial infarction. Our purpose was to investigate S100A6 gene therapy for acute myocardial ischemia‐reperfusion. Methods and ResultsWe first performed in vitro studies to examine the effects of S100A6 overexpression and knockdown in rat neonatal cardiomyocytes. S100A6 overexpression improved calcium transients and protected against apoptosis induced by hypoxia‐reoxygenation via enhanced calcineurin activity, whereas knockdown of S100A6 had detrimental effects. For in vivo studies, human S100A6 plasmid or empty plasmid was delivered to the left ventricular myocardium by ultrasound‐targeted microbubble destruction in Fischer‐344 rats 2 days prior to a 30‐minute ligation of the left anterior descending coronary artery followed by reperfusion. Control animals received no therapy. Pretreatment with S100A6 gene therapy yielded a survival advantage compared to empty‐plasmid and nontreated controls. S100A6‐pretreated animals had reduced infarct size and improved left ventricular systolic function, with less myocyte apoptosis, attenuated cardiac hypertrophy, and less cardiac fibrosis. ConclusionsS100A6 overexpression by ultrasound‐targeted microbubble destruction helps ameliorate myocardial ischemia‐reperfusion, resulting in lower mortality and improved left ventricular systolic function post–ischemia‐reperfusion via attenuation of apoptosis, reduction in cardiac hypertrophy, and reduced infarct size. Our results indicate that S100A6 is a potential therapeutic target for acute myocardial infarction.

Дана стаття присвячена літературному огляду методів візуалізації скелетних м’язів при м’язовій дистрофії Дюшенна. Проведено аналіз як історично значущих наукових робіт, так і останніх досягнень в галузі нейровізуалізації. У статті розглянуто такі методи, як ультразвукова діагностика, комп’ютерна та магнітно-резонансна томографія скелетних м’язів, а також магнітно-резонансна спектроскопія. Відзначено і обґрунтовано актуальність і перспективність неінвазивних, безпечних і об’єктивних методів оцінки скелетних м’язів при м’язовій дистрофії Дюшенна.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an intense fibrotic reaction termed tumor desmoplasia, which is in part responsible for its aggressiveness. Endothelial cells have been shown to display cellular plasticity in the form of endothelial-to-mesenchymal transition (EndMT) that serves as an important source of fibroblasts in pathological disorders, including cancer. Angiogenic co-receptor, neuropilin-1 (NRP- 1) actively binds TGFβ1, the primary mediator of EndMT and is involved in oncogenic processes like epithelial-to-mesenchymal transition (EMT). NRP-1 and TGFβ1 signaling have been shown to be aberrantly up-regulated in PDAC. We report herein a positive correlation between NRP-1 levels, EndMT and fibrosis in human PDAC xenografts. Loss of NRP-1 in HUVECs limited TGFβ1-induced EndMT as demonstrated by gain of endothelial and loss of mesenchymal markers, while maintaining endothelial cell architecture. Knockdown of NRP-1 down-regulated TGFβ canonical signaling (pSMAD2) and associated pro-fibrotic genes. Overexpression of NRP-1 exacerbated TGFβ1-induced EndMT and up-regulated TGFβ signaling and expression of pro-fibrotic genes. In vivo, loss of NRP-1 attenuated tumor perfusion and size, accompanied by reduction in EndMT and fibrosis. This study defines a previously unrecognized role of NRP-1 in regulating TGFβ1-induced EndMT and fibrosis, and advocates NRP-1 as a therapeutic target to reduce tumor fibrosis and PDAC progression.

Objective—MicroRNAs are involved in many critical functions, including angiogenesis. Ultrasound-targeted microbubble destruction (UTMD) is a noninvasive technique for targeted vascular transfection of plasmid DNA and may be well suited for proangiogenic microRNA delivery. We aimed to investigate UTMD of miR-126-3p for therapeutic angiogenesis in chronic ischemia. Approach and Results—The angiogenic potential of miR-126-3p was tested in human umbilical vein endothelial cells in vitro. UTMD of miR-126-3p was tested in vivo in Fischer-344 rats before and after chronic left femoral artery ligation, evaluating target knockdown, miR-126-3p and miR-126-5p expression, phosphorylated Tie2 levels, microvascular perfusion, and vessel density. In vitro, miR-126-3p–transfected human umbilical vein endothelial cells showed repression of sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2, negative regulators of vascular endothelial growth factor and angiopoietin-1 signaling, increased phosphorylated Tie2 mediated by knockdown of phosphatidylinositol-3-kinase regulatory subunit 2 and greater angiogenic potential mediated by both vascular endothelial growth factor/vascular endothelial growth factor R2 and angiopoietin-1/Tie2 effects. UTMD of miR-126-3p resulted in targeted vascular transfection, peaking early after delivery and lasting for >3 days, and resulting in inhibition of sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2, with minimal uptake in remote organs. Finally, UTMD of miR-126-3p to chronic ischemic hindlimb muscle resulted in improved perfusion, vessel density, enhanced arteriolar formation, pericyte coverage, and phosphorylated Tie2 levels, without affecting miR-126-5p or delta-like 1 homolog levels. Conclusions—UTMD of miR-126 results in improved tissue perfusion and vascular density in the setting of chronic ischemia by repressing sprouty-related protein-1 and phosphatidylinositol-3-kinase regulatory subunit 2 and enhancing vascular endothelial growth factor and angiopoietin-1 signaling, with no effect on miR-126-5p. UTMD is a promising platform for microRNA delivery, with applications for therapeutic angiogenesis.

Background: Left ventricular hypertrophy (LVH) is commonly found in chronic dialysis (CD) recipients, and is associated with impaired microvascular cardiac perfusion and heart failure. In response to LVH and cardiac ischemia, early outgrowth pro-angiogenic cellS(EPCs) mobilize from the bone marrow to facilitate angiogenesis and endothelial repair. In the general population, EPC number and function correlate inversely with cardiovascular risk. In end-stage renal disease (ESRD), EPC number and function are generally reduced. Objectives: To test whether left ventricular abnormalities retain their potent ability to promote EPC reparative responses in the setting of ESRD. Design: Cross-sectional study. Setting: St. Michael's Hospital, Toronto, Ontario, Canada. Patients: 47 prevalent chronic dialysis recipients. Measurements: (1) circulating CD34 + and CD133 + EPC number, (2) cultured EPC migratory ability, in vitro differentiation potential, and apoptosis rate, and (3) cardiac magnetic resonance-measured LV mass, volume and ejection fraction. Methods: Bivariate correlation analysis was performed with Spearman's rho test. Results: Of the 47 patients (mean age: 54 ± 13 years), the mean delivered urea reduction was 74 ± 10 %. Mean LV mass was 123 ± 38 g. Circulating CD34 + and CD133 + EPCs represented 0.14 % (IQR: 0.05 – 0.29 %) and 0.05 % (IQR: 0.01 – 0.10 %) of peripheral blood mononuclear cells. There were no significant correlations between any EPC parameter and measures of LV mass or ejection fraction. Limitations: Lack of a non-ESRD control population, and the inability to measure all parameters of EPC function due to limitations in blood sampling. Our inability to measure cardiac VEGF expression prevented an assessment of changes in cardiac EPC mobilization signals. Conclusions: These data suggest that in ESRD, the reparative EPC response to cardiac hypertrophy may be blunted. Further investigation of the effects of uremia on EPC physiology and its relationship to cardiac injury are required.

Ultrasound-targeted microbubble destruction (UTMD) is a non-invasive technique for non-viral gene delivery. Previously, we have tested UTMD for pro-angiogenic gene delivery using plasmids in rodent models of peripheral arterial disease (PAD). More recently, we have transitioned to minicircle DNA vectors over plasmids, due to longer lasting, greater gene expression. The Myocardial Infarction prone Watanabe heritable hyperlipidemic (WHHLMI) rabbit is a larger animal model of hypercholesterolemia that develops atherosclerotic plaque, and are an ideal pre-clinical model for testing UTMD of VEGF minicircles for PAD. WHHLMI rabbits (University of Kobe, Japan) underwent left femoral artery ligation and stripping (n=36). At day 3, week4, week8 and week12 post-ligation, microvascular blood flow (MBF) was assessed by contrast-enhanced ultrasound (CEU). UTMD of 1mg mini-circle-VEGF (MC-VEGF) (n=18) or 1mg empty vector (n=6) was performed at week4, with control animals (n=18) receiving no treatment. Angiography was performed at end study. PCR analysis was performed to assay gene expression. Total cholesterol (mmol/L) (10.4 ± 2.4 versus 0.7 ± 0.1) and triglycerides (mmol/L) (4.3 ± 0.1 versus 0.5 ± 0.1) were elevated in WHHLMI rabbits vs control New Zealand White rabbits. Normalized CEU perfusion of the ischemic hindlimb muscle at week8 and week12 showed a significant increase in MC-VEGF treatment versus control. (week8; 0.0097 ± 0.001 versus 0.0053 ± 0.001; p < 0.01, week12; 0.0059 ± 0.001 versus 0.0011 ± 0.0006; p < 0.01), with a late reduction in muscle perfusion seen in control animals. UTMD of empty vector MC showed no effect. Angiography showed poor collateralization in control animals, while MC-VEGF UTMD-treated animals had increased collateral vessel formation and more complete reconstitution of the distal arterial bed. Early (day3) PCR showed robust VEGF gene expression in MC-VEGF treated animals. UTMD of MC-VEGF results in significant improvement in tissue perfusion, at both early and late time points, in a severe PAD model in a clinically relevant WHHMMI rabbit model. Our study shows the potential of UTMD as an effective and safe gene delivery strategy to treat ischemic PAD in patients.

Data suggests that microRNAs (miRNAs) are essential regulators of gene expression. We have previously shown that over-expression of S100A6 ameliorates myocardial ischemia/reperfusion (I/R) injury. We now sought to determine the miRNAs involved in the regulation of S100A6 expression in cardiac I/R injury, as a means to discover novel miRNA targets, identifying miR-495 as a potential novel therapeutic target to prevent myocardial I/R injury. We applied miRNA microarray chip analysis to S100A6-overexpresssing and S100A6-knockdown neonatal rat ventricular myocytes (NRVM), with miRNAs showing significant expressional alteration then verified using one-step real-time PCR. Among the potential differentially regulated miRNAs, ten miRNAs were selected. These miRNAs were transfected into NRVMs to confirm the regulatory effect on S100A6 gene expression, with miR-495 identified as a strong target. We then transfected cultured NRVMs and human umbilical vein endothelial cells (HUVECs) with miR-495 (Lipofectamine RNAiMAX). Cells were exposed to hypoxia/re-oxygenation (H/R) injury (via hypoxic chamber) in vitro to simulate myocardial I/R. H/R-induced apoptosis was measured by Fluorescence Activated Cell Sorting (FACS) and expression of miR-495 and S100A6 were quantified by real-time PCR. Transfected HUVECs underwent matrigel angiogenesis assays. The expression of HIF-1α and mTOR were also quantified in the cultured cells to explore their participation in underlying pathways. miR-495 was differentially regulated by S100A6 over-expression and knockdown, while transfection of miR-495 significantly upregulated S100A6 gene expression. FACS analysis revealed that over-expression of miR-495 inhibited early and late apoptosis in cardiomyocytes and HUVECs when compared to the scrambled miRNA transfected cells (NRVMs early apoptosis: 1.3±0.1% vs. 5.3±0.4%, p<0.05) (NRVMs late apoptosis: 9.7±0.7% vs. 15.3±1.6%, p<0.05) (HUVECs early apoptosis: 7.3±0.4% vs. 9.1±1.1%, p<0.05) (HUVECs late apoptosis: 6.6±1.6% vs. 9.6±0.4%, p<0.05). In vitro matrigel angiogenesis assay indicated increased tube formation and number of nodes in miR-495 transfected HUVECs compared to non-transfected cells (tubes:15.8±0.6 vs. 12.8±0.8, p<0.05), (nodes:18.8±0.8 vs. 14.2±1.3, p<0.05). miR-495 transfected cardiomyocytes showed up-regulation of mTOR post H/R and up-regulation of HIF-1α under normoxic condition compared to the non-transfected cells, suggesting a contribution to miR-495 effect (mTOR post H/R: 1.21±0.12 vs. 0.80±0.01, p<0.05) (HIF1-α expression under normoxic condition: 1.24±0.03 vs. 1.0±0.04, p<0.01). We show that miR-495 transfection in NRVMs and HUVECs leads to an upregulation of S100A6, a pro-angiogenic effect, as well as up-regulation of HIF-1α and mTOR, both involved in cell survival and inflammatory response pathways following hypoxic stress. Our data supports miR-495 as a potential therapeutic target for cardiac I/R injury.

The endothelial specific microRNA-126 (miR-126) regulates angiogenesis primarily through the VEGFR-2 axis by blocking endogenous inhibitors of VEGF; Sprouty-related protein-1 (SPRED1) and phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2). We hypothesized that miR-126 transfection contributes to neovascularization through VEGFR-2 and Tie2 pathway at early and late stages in chronic ischemia.

Ultrasound-mediated gene delivery (UMGD) is a non-invasive transfection technique using gene-bearing microbubbles and focused high power ultrasound, and has been used primarily to deliver plasmid DNA. Minicircle-DNA is a novel gene vector, which has recently been shown to have an improved and prolonged transfection, compared to conventional plasmid-DNA. We hypothesized that UMGD of minicircle-DNA would demonstrate a more potent and persistent gene expression compared to conventional plasmid-DNA. In vitro, HUVECs, fibroblasts (3T3) and neonatal cardiomyocytes were transfected with molar equivalents of GFP-minicircle and GFP-plasmid by electroporation. GFP expression was evaluated by RT-PCR and fluorescent microscopy for 28 days. Next, we performed a comparison of bubble binding capacities of minicircle-DNA and conventional plasmid to cationic lipid microbubbles. In vivo, for UMGD to the left proximal hind-limb adductor muscle, 500μg and 214μg of GFP-plasmid and GFP-minicircle respectively were charge-coupled with 1x109 cationic microbubbles and delivered via UMGD into Sprague-Dawley rats (n=30). The transfection efficacy was followed for 28 days and measured by RT-PCR and immunohistochemistry. In vitro results showed greater GFP expression by minicircle-DNA across all cell lines, with transfection efficiency at 7-10 fold compared to plasmid-DNA. Minicircle-DNA demonstrated a significantly greater binding capacity to cationic microbubbles compared to plasmid-DNA. For plasmid DNA, binding saturation was reached at 250-500μg resulting in ∼6,000 plasmid copies per microbubble. For minicircle, binding saturation was reached at 125-250μg resulting in ∼20,000 minicircle copies per microbubble, suggesting a higher bubble binding efficiency. In vivo results showed higher GFP levels as early as 6 hours post minicircle UMGD, demonstrating minicircle to be a faster acting therapeutic agent over conventional plasmid. A significantly greater expression of GFP was also evident at day 28 in minicircle UMGD-treated group, proving minicircle-DNA to be a better choice for long-term gene expression. In summary, UMGD using minicircle-DNA results in more rapid and sustained transfection compared to conventional plasmid-DNA. Given this finding, minicircle vectors may allow for lower transmitted ultrasound power to be used for UMGD, potentially enhancing clinical applicability.

Background: Perlecan is a heparan sulfate proteoglycan (HSPG) constituent of the extracellular matrix with roles in cell growth, differentiation, and angiogenesis. The role of the HS side chains in regulating in vivo angiogenesis after hind-limb ischemia is unknown.Methods: Heparan sulfate (HS)-deficient perlecan (Hspg2(Delta 3/Delta 3)) mice (n = 35), containing normal perlecan core protein but deficient in HS side chains, and wild-type (n = 33) littermates underwent surgical induction of hind-limb ischemia. Laser Doppler perfusion imaging (LDPI) and contrast-enhanced ultrasonography (CEU) provided serial assessment of hind-limb perfusion. Harvested muscles underwent immunostaining for endothelial cell density (CD31), real-time reverse transcription polymerase chain reaction RT-PCR for vascular endothelial growth factor (VEGF) mRNA expression and western blot analysis for VEGF and fibroblast growth factor (FGF) 2 protein expression at days 2 and 28.Results: Serial LDPI showed significantly greater perfusion recovery in ischemic limbs of wild-type compared with Hspg2(Delta 3/Delta 3) mice. CEU showed that normalized microvascular perfusion was increased in wild-type compared with Hspg2(Delta 3/Delta 3) mice at day 28 (0.67 +/- 0.12 vs 0.26 +/- 0.08; P = 0.001). CD31-positive cell counts were significantly higher in wild-type compared with Hspg2(Delta 3/Delta 3) mice on day 28 (122 +/- 30 cells vs 84 +/- 34 cells per high-power field [HPF]; P < 0.05). Endogenous VEGF mRNA expression (P < 0.05) and VEGF protein expression (P < 0.002) were significantly decreased in the ischemic limbs of Hspg2(Delta 3/Delta 3) mice compared with wild-type mice at day 2 and day 28, respectively. FGF2 protein expression showed no significant differences.Conclusions: These results suggest that the HS side chains in perlecan are important mediators of the angiogenic response to ischemia through a mechanism that involves upregulation of VEGF expression.

Background: MicroRNA (miR)-126 is an endothelial cell specific microRNA that regulates angiogenesis by blocking Sprouty-related protein (SPRED1) and phosphoinositol-3 kinase (PI3K) two endogenous i...

Background: Ultrasound-targeted microbubble destruction (UTMD) is a non-invasive gene transfection technique using carrier microbubbles and targeted high power ultrasound, primarily used to deliver plasmid DNA (pDNA). Minicircle-DNA (mcDNA) is a novel gene vector, which has recently been shown to have an improved and persistent transfection, compared to conventional pDNA. Hypothesis: We hypothesized that UTMD of mcDNA in a hind-limb model would exhibit a more potent and prolonged gene expression compared to conventional pDNA. Methods: In vitro , HUVECs, fibroblasts (3T3) and neonatal cardiomyocytes were transfected with molar equivalents of GFP-minicircle and GFP-plasmid. GFP expression was measured by RT-PCR and fluorescent microscopy for 28 days. We then performed a comparison of bubble binding capacities of both vectors to cationic lipid microbubbles. In vivo , for UTMD to the left proximal hind-limb adductor muscle, 500μg and 214μg of GFP-plasmid and GFP-minicircle respectively were charge-coupled with 1x10 9 cationic microbubbles and delivered via UMGD into Sprague-Dawley rats (n=30). The animals were followed for 28 days with GFP measured by RT-PCR and immunohistochemistry. Results: In vitro results showed greater GFP expression by mcDNA across all cell lines, with 7-10 fold transfection efficacy compared to pDNA. mcDNA demonstrated greater binding capacity to cationic microbubbles compared to pDNA. For plasmid and minicircle DNA, binding saturations were reached at ~6000 copies per microbubble, and ~20000 copies per microbubble respectively, suggesting a higher minicircle bubble binding efficiency. In vivo results showed higher GFP levels as early as 6 hours post minicircle UTMD, demonstrating minicircle to be a faster acting therapeutic agent over conventional plasmid. A significantly greater (p<0.01) expression of GFP was also evident at day 28 in minicircle UTMD-treated group, proving mcDNA to be a better choice for longer-term gene expression. Conclusions: In summary, UTMD using mcDNA results in more rapid and sustained transfection compared to conventional pDNA, and may be a more effective vector for translational studies of UTMD.

Introduction: The endothelial specific microRNA-126 (miR-126) regulates angiogenesis primarily by blocking endogenous inhibitors of VEGF, Sprouty-related protein and phosphoinositol-3 kinase regulatory subunit 2. We hypothesized that ultrasound-mediated delivery of miR-126 contributes to neovascularization through VEGFR-2 and Tie2 pathway at an early and late stage respectively in chronic ischemia. Methods: F-344 rats (n=4) were delivered with fluorescently labeled miR by ultrasound-mediated gene delivery (UMGD) to determine the miR cellular localization. Unilateral hindlimb ischemia was created by left femoral artery ligation (n=92). At day 14 post-ligation, microvascular blood flow (MBF) was assessed by contrast-enhanced ultrasound (CEU). UMGD of miR-126 (n=35) or scrambled miR (n=25) was performed, with control animals (n=32) receiving no treatment. Perfusion was re-assessed and Tie2 and pTie2 levels were quantified by western blotting at day 28. For in vitro studies, human umbilical vascular endotheli...

Diabetes mellitus increases the risk of developing cardiovascular diseases and is associated with macro and microvascular complications. Various strategies to promote therapeutic angiogenesis in ischemic tissue have been investigated, including delivery of pro-angiogenic growth factors and the genes that encode them, but results have been disappointing. Risk factors, such as diabetes, may adversely impact the response to angiogenic strategies and limit efficacy in vivo. We sought to examine the revascularization effect of multiple pro-angiogenic genes on chronic hindlimb ischemia in both non-diabetic and diabetic mouse models. Spontaneously type II diabetic male mice (db/db; with C57/Bl6 genetic Background) and their control (db/m) between 8- and 10-week-old were studied. Unilateral hindlimb ischemia was induced in all animals by ligation and stripping of the left femoral artery. For gene delivery, plasmids containing pro-angiogenic genes were administrated intramuscularly directly into the ischemic distal hindlimb muscle, below the ligation site at 4 weeks (for early delivery) and at 6 weeks (for late delivery) after ligation procedure. Gene combinations included vascular endothelial growth factor (VEGF) and angiopoietin 1, 2 (Ang-1, 2). Relative blood flow to the foot was measured under standardized conditions by laser Doppler perfusion imaging (LDPI). Perfusion measurements were performed on postoperative days 1, 3, 7, week 2, 3, 4, 5, 6, 7 and 8. The perfusion ratio (occluded- to-non-occluded leg) was calculated for each animal. Animals from all groups were sacrificed at various times after gene delivery for tissue sampling (PCR, westerns, immunohistochemistry). Diabetic mice showed reduced ability to induce angiogenesis following ischemia compared to non-diabetic mice. In response to VEGF gene therapy, non-diabetic mice responded with greater neovascularization compared to diabetic mice. Ang-1 mono-therapy promoted an angiogenic response in diabetic mice, but not in non-diabetic mice. Ang-2 mono-therapy did not promote angiogenesis in either model. Temporally separated delivery of VEGF (early) and Ang-1 (late) promoted more neovascularization than any single gene therapy. Delivery of VEGF and Ang-2 (early) and Ang-1 (late) showed the greatest angiogenic response in both diabetic and non-diabetic mice. Diabetic mice show a reduced angiogenic response to VEGF and increased angiogenic response to Ang-1 in compared to non-diabetic mice. Temporally delivery of VEGF, Ang-2 and Ang-1 gene combination resulted a greater perfusion recovery and a greater angiogenic response in ischemic hindlimb skeletal muscle, compared to single gene or multiple gene combinations without temporal separation in both diabetic and non-diabetic settings.

BackgroundWe have previously shown that pre-delivery of S100A6 gene, an EF-hand Ca2+-binding protein, by ultrasound-targeted microbubble destruction (UTMD) results in lower mortality and improved LV systolic function after cardiac infarction-reperfusion (I/R). The S100A6 therapeutic effect on myocardial I/R was due in part to its anti-apoptotic effects and regulatory function on intracellular calcium cycling. Here we hypothesize that S100A6 therapeutic effects may also involve other pathways including angiogenesis and hypertrophic responses.MethodsUsing UTMD, we pre-delivered GFP-tagged human-S100A6 plasmid and empty plasmid to the LV in 10-12 weeks old Fischer- 344 male rats, 2 days prior to LAD-ligation (30 min) and reperfusion, with control animals receiving no UTMD. We measured the ratio of heart weight to tibia length at day 28 post I/R. Cardiomyocyte size was measured off haemotoxylin-eosin stained sections. Relative expression of βMHC and ANF were studied in the infarct and non-infarct regions of myocardium 28 days post I/R. Masson's Trichrome staining was performed to detect collagen fibers in the infarct zones. Myocardial contrast echo (MCE) was performed at day 28 to measure myocardial blood flow in the infarct and peri-infarct regions. For in vitro studies, human umbulical vein endothelial cells (HUVECs) were transduced with either adenoviral packaged GFP-tagged human-S100A6 plasmid, empty plasmid or rat S100A6-shRNA. Non-transduced cells served as additional controls. Matrigel tube formation assay was performed to observe the effects of S100A6 on capillary-like network formation.ResultsS100A6-treated hearts after cardiac I/R had significantly lower heart weight to tibia length (HW/TL) ratio, lower expression of β MHC and ANF in the infarct and non-infarct regions of myocardium and smaller cardiomyocyte size compared to controls at day 28. Extent of fibrosis/infarct size at day 28 was significantly lower in S100A6 pre-treated animals compared to empty plasmid and non-treated controls. MCE-derived myocardial blood flow at day 28 post ischemia/reperfusion in the risk area (infarct and peri-infarct regions) normalized to the remote non-infarct regions was significantly higher in the S100A6 treated group confirmed with IHC staining showing increased vessel density there. S100A6 over-expressing HUVECs formed significantly greater number of nodes and tubes compared to null-transduced and non-transduced controls after 3 hours.ConclusionS100A6 overexpression results in reduced infarct size, not only by prevention of cardiomyocyte apoptosis (based on our previous data), but also via prevention of late fibrosis, enhancement of angiogenesis within the infarct and peri-infarct regions and attenuation of myocyte hypertrophy after acute infarction-reperfusion. BackgroundWe have previously shown that pre-delivery of S100A6 gene, an EF-hand Ca2+-binding protein, by ultrasound-targeted microbubble destruction (UTMD) results in lower mortality and improved LV systolic function after cardiac infarction-reperfusion (I/R). The S100A6 therapeutic effect on myocardial I/R was due in part to its anti-apoptotic effects and regulatory function on intracellular calcium cycling. Here we hypothesize that S100A6 therapeutic effects may also involve other pathways including angiogenesis and hypertrophic responses. We have previously shown that pre-delivery of S100A6 gene, an EF-hand Ca2+-binding protein, by ultrasound-targeted microbubble destruction (UTMD) results in lower mortality and improved LV systolic function after cardiac infarction-reperfusion (I/R). The S100A6 therapeutic effect on myocardial I/R was due in part to its anti-apoptotic effects and regulatory function on intracellular calcium cycling. Here we hypothesize that S100A6 therapeutic effects may also involve other pathways including angiogenesis and hypertrophic responses. MethodsUsing UTMD, we pre-delivered GFP-tagged human-S100A6 plasmid and empty plasmid to the LV in 10-12 weeks old Fischer- 344 male rats, 2 days prior to LAD-ligation (30 min) and reperfusion, with control animals receiving no UTMD. We measured the ratio of heart weight to tibia length at day 28 post I/R. Cardiomyocyte size was measured off haemotoxylin-eosin stained sections. Relative expression of βMHC and ANF were studied in the infarct and non-infarct regions of myocardium 28 days post I/R. Masson's Trichrome staining was performed to detect collagen fibers in the infarct zones. Myocardial contrast echo (MCE) was performed at day 28 to measure myocardial blood flow in the infarct and peri-infarct regions. For in vitro studies, human umbulical vein endothelial cells (HUVECs) were transduced with either adenoviral packaged GFP-tagged human-S100A6 plasmid, empty plasmid or rat S100A6-shRNA. Non-transduced cells served as additional controls. Matrigel tube formation assay was performed to observe the effects of S100A6 on capillary-like network formation. Using UTMD, we pre-delivered GFP-tagged human-S100A6 plasmid and empty plasmid to the LV in 10-12 weeks old Fischer- 344 male rats, 2 days prior to LAD-ligation (30 min) and reperfusion, with control animals receiving no UTMD. We measured the ratio of heart weight to tibia length at day 28 post I/R. Cardiomyocyte size was measured off haemotoxylin-eosin stained sections. Relative expression of βMHC and ANF were studied in the infarct and non-infarct regions of myocardium 28 days post I/R. Masson's Trichrome staining was performed to detect collagen fibers in the infarct zones. Myocardial contrast echo (MCE) was performed at day 28 to measure myocardial blood flow in the infarct and peri-infarct regions. For in vitro studies, human umbulical vein endothelial cells (HUVECs) were transduced with either adenoviral packaged GFP-tagged human-S100A6 plasmid, empty plasmid or rat S100A6-shRNA. Non-transduced cells served as additional controls. Matrigel tube formation assay was performed to observe the effects of S100A6 on capillary-like network formation. ResultsS100A6-treated hearts after cardiac I/R had significantly lower heart weight to tibia length (HW/TL) ratio, lower expression of β MHC and ANF in the infarct and non-infarct regions of myocardium and smaller cardiomyocyte size compared to controls at day 28. Extent of fibrosis/infarct size at day 28 was significantly lower in S100A6 pre-treated animals compared to empty plasmid and non-treated controls. MCE-derived myocardial blood flow at day 28 post ischemia/reperfusion in the risk area (infarct and peri-infarct regions) normalized to the remote non-infarct regions was significantly higher in the S100A6 treated group confirmed with IHC staining showing increased vessel density there. S100A6 over-expressing HUVECs formed significantly greater number of nodes and tubes compared to null-transduced and non-transduced controls after 3 hours. S100A6-treated hearts after cardiac I/R had significantly lower heart weight to tibia length (HW/TL) ratio, lower expression of β MHC and ANF in the infarct and non-infarct regions of myocardium and smaller cardiomyocyte size compared to controls at day 28. Extent of fibrosis/infarct size at day 28 was significantly lower in S100A6 pre-treated animals compared to empty plasmid and non-treated controls. MCE-derived myocardial blood flow at day 28 post ischemia/reperfusion in the risk area (infarct and peri-infarct regions) normalized to the remote non-infarct regions was significantly higher in the S100A6 treated group confirmed with IHC staining showing increased vessel density there. S100A6 over-expressing HUVECs formed significantly greater number of nodes and tubes compared to null-transduced and non-transduced controls after 3 hours. ConclusionS100A6 overexpression results in reduced infarct size, not only by prevention of cardiomyocyte apoptosis (based on our previous data), but also via prevention of late fibrosis, enhancement of angiogenesis within the infarct and peri-infarct regions and attenuation of myocyte hypertrophy after acute infarction-reperfusion. S100A6 overexpression results in reduced infarct size, not only by prevention of cardiomyocyte apoptosis (based on our previous data), but also via prevention of late fibrosis, enhancement of angiogenesis within the infarct and peri-infarct regions and attenuation of myocyte hypertrophy after acute infarction-reperfusion.

Background: Diabetes mellitus is a chronic metabolic disease associated with various vascular complications. Various strategies to promote therapeutic angiogenesis in ischemic tissue including delivery of pro-angiogenic growth factors have shown disappointing results. Risk factors, such as diabetes, may adversely impact the response to angiogenic strategies and limit efficacy in vivo. We assessed the hypothesis that temporally separated delivery of multiple pro-angiogenic growth factors will improve the efficacy of the angiogenic response in the setting of both diabetes and non-diabetes in vitro. Methods: 96-well tissue culture plates were coated with 60 μL growth factor reduced Matrigel. After gelation, non-diabetic human dermal microvascular endothelial cells (HMVECs) and type-II diabetic cells (D-HMVECs) were plated at 1x104 cells/well with 100 μL basal media EBM-2 with 0.1% bovine serum, and incubated at 37°C. Combinations of growth factors including recombinant human VEGF (50 ng/mL), angiopoietin (Ang)-1 and -2 (250 ng/mL) were administrated at start of the assay and/or at 6 hours into the assay. Images were taken at 3, 6, 9, 12, 18 and 24 hours and used for tube formation quantification by Inverted Microscope at 10x magnification. Results: In untreated control, D-HVECs showed reduced tube formation in compared to HMVECs (n=12; p<0.001). Growth factors combination ‘VEGF and Ang-2 at start of the assay and Ang-1 at 6 hours showed the most significantly increased of tube formation when compared to single growth factor (to VEGF, Ang-1, 2; p<0.001) or growth factors combination without temporally separated delivery (to VEGF/Ang-1, VEGF/Ang-2; p<0.001) in both HMVEC and D-HMVEC. Conclusion: Diabetic endothelial cells showed impaired angiogenesis compared to non-diabetic endothelial cells. Combinations of temporally separated pro-angiogenic growth factors were able to optimize the angiogenic effects and yield the most significant efficacy in the setting of both diabetes and non-diabetes. Future studies will focus on in vivo temporally separated combinations of multiple growth factors.

We have previously shown that UTMD of plasmid-S100A6, an EF-hand Ca2+-binding protein, 2 days prior to ligation/reperfusion attenuates myocardial infarction-reperfusion (I/R) injury, resulting in lower mortality and improved LV systolic function. The next step towards clinical translation is the development of UTMD techniques for effective delivery at the time of coronary reperfusion. Preliminary experiments with UTMD of minicircle DNA suggest more rapid and sustained transfection, and thus may be a more effective vector for UTMD in acute I/R. Hypothesis: We hypothesize that UTMD of minicircle-S100A6 (MC-S100A6), a very small episomal DNA vector, will lead to smaller infarct size and greater LV systolic function after myocardial I/R. Male Fischer rats (n=20; 150-200 g), underwent I/R induction surgery by a 30-minute ligation of the left anterior descending coronary artery (LAD) followed by reperfusion. MC-S100A6 (214 mcg+1x106 cationic microbubbles) was delivered by UTMD to the left ventricle of animals immediately after coronary reperfusion. Control animals received no therapy. Animals were followed by serial echocardiography to evaluate LV systolic function and circumferential extent of akinesis at various time points until day 28. Subgroups of animals were sacrificed at earlier time points to collect tissue for PCR and Western blotting. We observed robust expression of exogenous S100A6 in all regions of the heart with maximal transfection in the infarct and per-infarct (anterior) regions at day 3 post-delivery. Western blot data of human S100A6 protein was consistent with PCR results. Echo data from control untreated and MC-S100A6 UTMD-treated rats after I/R injury showed that animals treated with UTMD of MC-S100A6 had significantly improved fractional shortening (33.8% vs. 20.9%, P<0.05, day 28) and LVEF (59.7% vs. 45.5%, P<0.05, day 28), with less circumferential extent of akinesis (24.1% vs. 31.3%, P<0.05, day 28) compared to non-treated controls. UTMD of minicircle-S100A6 immediately after coronary reperfusion results in targeted transfection in the infarct and peri-infarct regions, leading to improved LV systolic function and less regional wall motion abnormalities, after acute myocardial infarction-reperfusion.

Aims The aim of this study was to investigate anti-apoptotic gene therapy using ultrasound-mediated plasmid delivery of survivin, an inhibitor of apoptosis protein, to prevent apoptosis and to attenuate left ventricular (LV) systolic dysfunction in a model of heart failure induced by doxorubicin.Methods and results Effect of survivin transduction was investigated in vitro in rat cardiomyoblasts. After survivin transduction, survivin protein was detected in cell culture supernate confirming secretion of extracellular survivin. Under doxorubicin stimulation, survivin-transduced cells had significantly reduced apoptosis; however, incubation with survivin-conditioned media also showed reduced apoptosis that was absent with null-conditioned media. Doxorubicin-induced cardiomyopathy was established in Fischer rats. Subsets of animals underwent ultrasound-mediated survivin gene delivery or empty vector gene delivery at Week 3. Control rats received doxorubicin alone. Animals were studied using PCR, immunohistochemistry, echocardiography, and invasive haemodynamic studies out to Week 6. By Week 6, LV% fractional shortening by echocardiography and systolic function by pressure-volume loops were greater in survivin treated when compared with control-and empty-treated animals. There was reduced apoptosis by TUNEL and caspase activity in survivin-treated animals compared with control and empty treated at Week 4, with reduced interstitial fibrosis at Week 6.Conclusion Survivin gene therapy can attenuate the progression of LV systolic dysfunction in doxorubicin cardiomyopathy. This effect can be attributed to decreased myocyte apoptosis and prevention of maladaptive LV remodelling, by both direct myocyte transfection and potentially by paracrine mechanisms.