Cardiac Overexpression of S100A6 Attenuates Cardiomyocyte Apoptosis and Reduces Infarct Size After Myocardial Ischemia-Reperfusion.

BACKGROUND: Cardiomyocyte-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 RESULTS: We 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. CONCLUSIONS: S100A6 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.

Novel regulatory role of neuropilin-1 in endothelial-to-mesenchymal transition and fibrosis in pancreatic ductal adenocarcinoma.

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 TGFß1, the primary mediator of EndMT and is involved in oncogenic processes like epithelial-to-mesenchymal transition (EMT). NRP-1 and TGFß1 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 TGFß1-induced EndMT as demonstrated by gain of endothelial and loss of mesenchymal markers, while maintaining endothelial cell architecture. Knockdown of NRP-1 down-regulated TGFß canonical signaling (pSMAD2) and associated pro-fibrotic genes. Overexpression of NRP-1 exacerbated TGFß1-induced EndMT and up-regulated TGFß 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 TGFß1-induced EndMT and fibrosis, and advocates NRP-1 as a therapeutic target to reduce tumor fibrosis and PDAC progression.

Prospect of ultrasound-mediated gene delivery in cardiovascular applications.

INTRODUCTION: The field of regenerative medicine has evolved over the years, investigating gene and stem/progenitor cell therapies to help address the increasing burden of cardiovascular disease (CVD). While the lack of success of gene therapy in clinical trials has dampened enthusiasm, the search continues for a successful and translatable gene therapy strategy for CVD. Ultrasound-mediated gene delivery (UMGD) is a non-invasive technique for gene delivery that utilizes gene-bearing carrier microbubbles and high power ultrasound to facilitate transfection in vivo. Many pre-clinical studies have shown benefit in animal models of CVD, but this has yet to be translated to human applications. AREAS COVERED: In this review, the basic principles of UMGD will be examined along with an overview of pre-clinical studies to date in CVD, focusing on cardiac and vascular applications and key findings. In addition, the potential path to the clinical translation of UMGD is discussed. EXPERT OPINION: Ultrasound-mediated gene delivery holds promise as a non-invasive technique for gene delivery in CVD, with the ability to deliver multiple genes with repeated deliveries over time. If the substantial hurdles to clinical translation can be overcome, UMGD may prove to be a key aspect in the success of cardiovascular gene therapy in the future.

Therapeutic Angiogenesis by Ultrasound-Mediated MicroRNA-126-3p Delivery.

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.

A modified portfolio diet complements medical management to reduce cardiovascular risk factors in diabetic patients with coronary artery disease.

BACKGROUND & AIMS: Secondary prevention can improve outcomes in high risk patients. This study investigated the magnitude of cardiovascular risk reduction associated with consumption of a modified portfolio diet in parallel with medical management. DESIGN: 30 patients with type II diabetes, 6 weeks post bypass surgery received dietary counseling on a Modified Portfolio Diet (MPD) (low fat, 8 g/1000 kcal viscous fibres, 17 g/1000 kcal soy protein and 22 g/1000 kcal almonds). Lipid profiles, endothelial function and markers of glycemic control, oxidative stress and inflammation were measured at baseline and following two and four weeks of intervention. Seven patients with no diet therapy served as time controls. RESULTS: Consumption of the MPD resulted in a 19% relative reduction in LDL (1.9 ± 0.8 vs 1.6 ± 0.6 mmol/L, p < 0.001) with no change in HDL cholesterol. Homocysteine levels dropped significantly (10.1 ± 2.7 vs 7.9 ± 4 µmol/L, p = 0.006) over the study period. Flow mediated dilatation increased significantly in treated patients (3.8 ± 3.8% to 6.5 ± 3.6%, p = 0.004) while remaining constant in controls (p = 0.6). Endothelial progenitor cells numbers (CD34+, CD 133+ and UEA-1+) increased significantly following MPD consumption (p < 0.02) with no difference in migratory capacity. In contrast, time controls showed no significant changes. CONCLUSION: Dietary intervention in medically managed, high risk patients resulted in important reductions in risk factors. Clinical Trials registry number NCT00462436.

Perlecan heparan sulfate proteoglycan is a critical determinant of angiogenesis in response to mouse hind-limb ischemia.

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(Δ3/Δ3)) 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(Δ3/Δ3) mice. CEU showed that normalized microvascular perfusion was increased in wild-type compared with Hspg2(Δ3/Δ3) 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(Δ3/Δ3) 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(Δ3/Δ3) 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.

Circulating angiogenic cell dysfunction in patients with hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant vascular disorder. Circulating angiogenic cells (CACs) play an important role in vascular repair and regeneration. This study was designed to examine the function of CACs derived from patients with HHT. Peripheral blood mononuclear cells (PBMNCs) isolated from patients with HHT and age- and gender-matched healthy volunteers were assessed for expression of CD34, CD133 and VEGF receptor 2 by flow cytometry. PBMNCs were cultured to procure early outgrowth CACs. Development of endothelial cell (EC) phenotype in CACs was analyzed by fluorescence microscopy. CAC apoptosis was assayed with Annexin V staining, and CAC migration assessed by a modified Boyden chamber assay. mRNA expression of endoglin (ENG), activin receptor-like kinase-1 (ACVLR1 or ALK1) and endothelial nitric oxide synthase (eNOS) in CACs was measured by real time RT-PCR. The percentage of CD34+ cells in PBMNCs from HHT patients was significantly higher than in PBMNCs of healthy controls. CACs derived from patients with HHT not only showed a significant reduction in EC-selective surface markers following 7-day culture, but also a significant increase in the rate of basal apoptosis and blunted migration in response to vascular endothelial growth factor and stromal cell-derived factor-1. CACs from HHT patients expressed significantly lower levels of ENG, ALK1 and eNOS mRNAs. In conclusion, CACs from patients with HHT exhibited various functional impairments, suggesting a reduced regenerative capacity of CACs to repair the vascular lesions seen in HHT patients.

Survivin gene therapy attenuates left ventricular systolic dysfunction in doxorubicin cardiomyopathy by reducing apoptosis and fibrosis.

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.

Optimization of Ultrasound-mediated Anti-angiogenic Cancer Gene Therapy.

Ultrasound-targeted microbubble destruction (UTMD) can be used to deliver silencing gene therapy to tumors. We hypothesized that UTMD would be effective in suppressing angiogenesis within tumors, and that modulation of the ultrasound pulsing intervals (PI) during UTMD would affect the magnitude of target knockdown. We performed UTMD of vascular endothelial growth factor receptor-2 (VEGFR2) short hairpin (sh)RNA plasmid in an heterotopic mammary adenocarcinoma model in rats, evaluating PIs of 2, 5, 10, and 20 seconds. We demonstrated that UTMD with a PI of 10 seconds resulted in the greatest knockdown of VEGFR2 by PCR, immunostaining, western blotting, smaller tumor volumes and perfused areas, and lower tumor microvascular blood volume (MBV) and flow by contrast-enhanced ultrasound (CEU) compared with UTMD-treated tumors at 2, 5, and 20 seconds, control tumors, tumors treated with intravenous shRNA plasmid and scrambled plasmid. CEU perfusion assessment using the therapeutic probe demonstrated that tumors were fully replenished with microbubbles within 10 seconds, but incompletely replenished at PI-2 and PI-5 seconds. In conclusion, for anti-VEGFR2 cancer gene therapy by UTMD, PI of 10 seconds results in higher target knockdown and a greater anti-angiogenic effect. Complete replenishment of tumor vasculature with silencing gene-bearing microbubbles in between destructive pulses of UTMD is required to maximize the efficacy of anti-angiogenic cancer gene therapy.Molecular Therapy - Nucleic Acids (2013) 2, e94; doi:10.1038/mtna.2013.20; published online 21 May 2013.

A direct comparison of endothelial progenitor cell dysfunction in rat metabolic syndrome and diabetes.

OBJECTIVES: Diabetes mellitus (DM) is associated with impairment of endothelial progenitor cells (EPCs), but the effects of metabolic syndrome (MS) on EPCs have been less well characterized. We hypothesized that in the presence of MS, the number and functionality of EPCs would be markedly reduced, and would be similar to DM. METHODS: Mononuclear cells were isolated from the bone-marrow (BM) and peripheral blood of lean Zucker, obese Zucker, a model of MS, and Zucker diabetic fatty rats. Cultured BM-EPCs underwent in vitro functional testing and the ability of BM-EPCs to promote neovascularization in vivo was assessed in a model of hindlimb ischemia in athymic mice. RESULTS: While circulating EPC numbers were similarly reduced in both MS and DM rats, BM-derived EPC numbers were less affected. In vitro testing of cultured BM-EPCs from obese Zucker demonstrated a marked reduction in EPC differentiation, a greater propensity to apoptosis, a reduced migratory response and matrigel tubule formation, similar to findings in Zucker diabetic fatty rats. When delivered to the ischemic hindlimb of athymic mice, the recovery of perfusion using both BM-EPCs from obese Zucker and Zucker diabetic fatty rats were diminished, as compared to lean Zuckers. CONCLUSION: In the presence of the MS, BM-derived EPCs develop marked functional impairment, resulting in severely reduced angiogenic capacity in vivo. Similar to DM, EPC dysfunction may play a prominent role in the pathogenesis of vascular complications in the MS, and may potentially limit the use of BM-derived EPCs for therapeutic angiogenesis.

Ultrasound-mediated vascular gene transfection by cavitation of endothelial-targeted cationic microbubbles.

OBJECTIVES: Ultrasound-mediated gene delivery can be amplified by acoustic disruption of microbubble carriers that undergo cavitation. We hypothesized that endothelial targeting of microbubbles bearing cDNA is feasible and, through optimizing proximity to the vessel wall, increases the efficacy of gene transfection. BACKGROUND: Contrast ultrasound-mediated gene delivery is a promising approach for site-specific gene therapy, although there are concerns with the reproducibility of this technique and the safety when using high-power ultrasound. METHODS: Cationic lipid-shelled decafluorobutane microbubbles bearing a targeting moiety were prepared and compared with nontargeted microbubbles. Microbubble targeting efficiency to endothelial adhesion molecules (P-selectin or intercellular adhesion molecule [ICAM]-1) was tested using in vitro flow chamber studies, intravital microscopy of tumor necrosis factor-alpha (TNF-α)-stimulated murine cremaster muscle, and targeted contrast ultrasound imaging of P-selectin in a model of murine limb ischemia. Ultrasound-mediated transfection of luciferase reporter plasmid charge coupled to microbubbles in the post-ischemic hindlimb muscle was assessed by in vivo optical imaging. RESULTS: Charge coupling of cDNA to the microbubble surface was not influenced by the presence of targeting ligand, and did not alter the cavitation properties of cationic microbubbles. In flow chamber studies, surface conjugation of cDNA did not affect attachment of targeted microbubbles at microvascular shear stresses (0.6 and 1.5 dyne/cm(2)). Attachment in vivo was also not affected by cDNA according to intravital microscopy observations of venular adhesion of ICAM-1-targeted microbubbles and by ultrasound molecular imaging of P-selectin-targeted microbubbles in the post-ischemic hindlimb in mice. Transfection at the site of high acoustic pressures (1.0 and 1.8 MPa) was similar for control and P-selectin-targeted microbubbles but was associated with vascular rupture and hemorrhage. At 0.6 MPa, there were no adverse bioeffects, and transfection was 5-fold greater with P-selectin-targeted microbubbles. CONCLUSIONS: We conclude that ultrasound-mediated transfection at safe acoustic pressures can be markedly augmented by endothelial juxtaposition.

Sustained improvement in perfusion and flow reserve after temporally separated delivery of vascular endothelial growth factor and angiopoietin-1 plasmid deoxyribonucleic acid.

OBJECTIVES: The aim of this study was to compare temporally separated vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-1 delivery with concomitant delivery or single VEGF delivery, for therapeutic angiogenesis in chronic ischemia. BACKGROUND: Single gene delivery of VEGF results in immature neovessels that ultimately regress. Endogenously, VEGF acts early to initiate angiogenesis, whereas Ang-1 acts later to induce vessel maturation. Timing VEGF and Ang-1 gene delivery to mimic endogenous angiogenesis might be more effective for sustained neovascularization. METHODS: Unilateral hindlimb ischemia was induced in 170 rats. Ultrasound-mediated gene delivery was performed with cationic microbubbles and plasmid deoxyribonucleic acid. Groups included VEGF at 2 weeks, VEGF/Ang-1 at 2 weeks, VEGF at 2 weeks with Ang-1 at 4 weeks, and untreated control subjects. At 2, 4, and 8 weeks after ligation, blood flow and flow reserve (FR) were assessed by contrast-enhanced ultrasound. Vascular density, organization, and supporting cell coverage were assessed by fluorescent microangiography and immunohistochemistry. RESULTS: In untreated control subjects, blood flow, FR, and vessel density remained reduced. The VEGF delivery improved flow and vessel density at 4 weeks; however, FR remained low, supporting cell coverage was poor, and flow and vessel density regressed by 8 weeks. The VEGF/Ang-1 co-delivery marginally increased flow and vessel density; however, FR and supporting cell coverage improved. After temporally separated VEGF and Ang-1 delivery, blood flow, vessel density, and FR increased and were sustained, with improved pericyte coverage at 8 weeks. CONCLUSIONS: In conclusion, temporally separated VEGF and Ang-1 gene therapy results in sustained and functional neovascularization.

Fluorescent microangiography is a novel and widely applicable technique for delineating the renal microvasculature.

Rarefaction of the renal microvasculature correlates with declining kidney function. However, current technologies commonly used for its evaluation are limited by their reliance on endothelial cell antigen expression and assessment in two dimensions. We set out to establish a widely applicable and unbiased optical sectioning method to enable three dimensional imaging and reconstruction of the renal microvessels based on their luminal filling. The kidneys of subtotally nephrectomized (SNx) rats and their sham-operated counterparts were subjected to either routine two-dimensional immunohistochemistry or the novel technique of fluorescent microangiography (FMA). The latter was achieved by perfusion of the kidney with an agarose suspension of fluorescent polystyrene microspheres followed by optical sectioning of 200 µm thick cross-sections using a confocal microscope. The fluorescent microangiography method enabled the three-dimensional reconstruction of virtual microvascular casts and confirmed a reduction in both glomerular and peritubular capillary density in the kidneys of SNx rats, despite an overall increase in glomerular volume. FMA is an uncomplicated technique for evaluating the renal microvasculature that circumvents many of the limitations imposed by conventional analysis of two-dimensional tissue sections.

Nocturnal hemodialysis is associated with restoration of early-outgrowth endothelial progenitor-like cell function.

BACKGROUND AND OBJECTIVES: Angiogenesis is a key response to tissue ischemia that may be impaired by uremia. Although early-outgrowth endothelial progenitor-like cells promote angiogenesis in the setting of normal renal function, cells from uremic patients are dysfunctional. When compared with conventional hemodialysis, it was hypothesized that nocturnal hemodialysis would improve the in vivo angiogenic activity of these cells in a well described model of ischemic vascular disease. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: Early-outgrowth endothelial progenitor-like cells were cultured from healthy controls (n = 5) and age- and gender-matched conventional hemodialysis (12 h/wk, n = 10) and nocturnal hemodialysis (30 to 50 h/wk, n = 9) patients. Cells (5 × 10(5)) or saline were injected into the ischemic hindlimb of athymic nude rats 1 day after left common iliac artery ligation. RESULTS: Although conventional dialysis cell injection had no effect versus saline, nocturnal hemodialysis and healthy control cell injection significantly improved ischemic hindlimb perfusion and capillary density. Nocturnal hemodialysis cell injection was also associated with significant increases in endogenous angiopoietin 1 expression in the ischemic hindlimb compared with saline and conventional dialysis cell injection. CONCLUSIONS: In contrast to a conventional dialytic regimen, nocturnal hemodialysis is associated with a significantly improved ability of early-outgrowth endothelial progenitor-like cells to promote angiogenesis and thus restore perfusion in a model of ischemic vascular disease.

Contrast ultrasound and targeted microbubbles: diagnostic and therapeutic applications for angiogenesis.

Angiogenesis represents the formation of new capillaries from existing vasculature, and as such plays a critical role in the response to ischemia in the setting of chronic coronary artery and peripheral vascular disease. Recent technological advances in non-invasive imaging modalities now allow the molecular imaging of angiogenesis. One such technique is contrast-enhanced ultrasound using microbubbles targeted against molecular markers of the angiogenic process. The ability to non-invasively image the angiogenic process would be useful in risk stratifying patients with arterial occlusive disease and would aid in the evaluation of new therapies to promote angiogenesis in ischemic cardiac and skeletal muscle. Furthermore, ultrasound technologies have also been developed that allow targeted angiogenic gene therapy using high-power ultrasound and DNA-bearing microbubbles. This review will focus specifically on recent advances in (1) contrast-enhanced ultrasound molecular imaging techniques for the evaluation of angiogenesis and (2) ultrasound-mediated gene delivery for therapeutic angiogenesis, techniques that have potential for translation to clinical practice.

Vascular gene transfer of SDF-1 promotes endothelial progenitor cell engraftment and enhances angiogenesis in ischemic muscle.

Gene therapy approaches to enhance endothelial progenitor cell (EPC) homing may augment cell engraftment to ischemic tissue and lead to a greater therapeutic response. Therefore, we assessed the effects of ultrasound-mediated (UM) transfection of the chemokine stromal cell-derived factor-1 (SDF-1) on homing and engraftment of intravenously administered EPCs and the subsequent angiogenic response in chronically ischemic skeletal muscle. Bone marrow-derived EPCs were isolated from donor Fisher 344 rats, cultured and labeled in preparation for injection into recipient animals via a jugular vein. Using a model of chronic hindlimb ischemia in rats, we demonstrated that UM destruction of intravenous carrier microbubbles loaded with SDF-1 plasmid DNA resulted in targeted transfection of the vascular endothelium within ischemic muscle and greater local engraftment of EPCs. The combination of SDF-1gene therapy and EPCs lead to the greatest increase in tissue perfusion and microvascular density within ischemic muscle, compared to no treatment or either monotherapy alone. Our results demonstrate that UM transfection of SDF-1 improves EPC targeting to chronically ischemic tissue, enhancing vascular engraftment and leading to a more robust neovascularization response.

Culture-modified bone marrow cells attenuate cardiac and renal injury in a chronic kidney disease rat model via a novel antifibrotic mechanism.

BACKGROUND: Most forms of chronic kidney disease are characterized by progressive renal and cardiac fibrosis leading to dysfunction. Preliminary evidence suggests that various bone marrow-derived cell populations have antifibrotic effects. In exploring the therapeutic potential of bone marrow derived cells in chronic cardio-renal disease, we examined the anti-fibrotic effects of bone marrow-derived culture modified cells (CMCs) and stromal cells (SCs). METHODOLOGY/PRINCIPAL FINDINGS: In vitro, CMC-conditioned medium, but not SC-conditioned medium, inhibited fibroblast collagen production and cell signalling in response to transforming growth factor-beta. The antifibrotic effects of CMCs and SCs were then evaluated in the 5/6 nephrectomy model of chronic cardio-renal disease. While intravascular infusion of 10(6) SCs had no effect, 10(6) CMCs reduced renal fibrosis compared to saline in the glomeruli (glomerulosclerosis index: 0.8+/-0.1 v 1.9+/-0.2 arbitrary units) and the tubulointersitium (% area type IV collagen: 1.2+/-0.3 v 8.4+/-2.0, p<0.05 for both). Similarly, 10(6) CMCs reduced cardiac fibrosis compared to saline (% area stained with picrosirius red: 3.2+/-0.3 v 5.1+/-0.4, p<0.05), whereas 10(6) SCs had no effect. Structural changes induced by CMC therapy were accompanied by improved function, as reflected by reductions in plasma creatinine (58+/-3 v 81+/-11 micromol/L), urinary protein excretion (9x/divided by 1 v 64x/divided by 1 mg/day), and diastolic cardiac stiffness (left ventricular end-diastolic pressure-volume relationship: 0.030+/-0.003 v 0.058+/-0.011 mm Hg/microL, p<0.05 for all). Despite substantial improvements in structure and function, only rare CMCs were present in the kidney and heart, whereas abundant CMCs were detected in the liver and spleen. CONCLUSIONS/SIGNIFICANCE: Together, these findings provide the first evidence suggesting that CMCs, but not SCs, exert a protective action in cardio-renal disease and that these effects may be mediated by the secretion of diffusible anti-fibrotic factor(s).

Comparison of gene delivery techniques for therapeutic angiogenesis ultrasound-mediated destruction of carrier microbubbles versus direct intramuscular injection.

OBJECTIVES: This study was designed to compare the efficacy of angiogenic gene delivery by ultrasound-mediated (UM) destruction of intravenous carrier microbubbles to direct intramuscular (IM) injections. BACKGROUND: Current trials of gene therapy for angiogenesis remain limited by suboptimal, invasive delivery techniques. METHODS: Hind-limb ischemia was produced by iliac artery ligation in 99 rats. In 32 rats, UM delivery of green fluorescent protein (GFP)/vascular endothelial growth factor-165 (VEGF(165)) plasmid deoxyribonucleic acid was performed. Thirty-five animals received IM injections of VEGF(165)/GFP plasmid. Remaining rats received no treatment. Before delivery (day 14 after ligation) and at days 17, 21, and 28 and week 8 after ligation, microvascular blood volume and microvascular blood flow to the proximal hind limbs were assessed by contrast-enhanced ultrasound (n = 8 per group). Total transfection was assessed by reverse transcriptase-polymerase chain reaction, and localization of transfection was determined by immunohistochemistry. RESULTS: By day 28, both IM and UM delivery of VEGF(165) produced significant increases in microvascular blood volume and microvascular blood flow. Whereas increases in microvascular blood volume were similar between treatment groups, microvascular blood flow was greater (p < 0.005) in UM-treated animals as compared with IM-treated animals, persisting to week 8. The VEGF(165)/GFP messenger ribonucleic acid expression was greater (p < 0.05) for IM-treated animals. A strong GFP signal was detected for both groups and was localized to focal perivascular regions and myocytes around injection sites for IM and to the vascular endothelium of arterioles/capillaries in a wider distribution for UM delivery. CONCLUSIONS: Despite lower transfection levels, UM delivery of VEGF(165) is as effective as IM injections. The UM delivery results in directed vascular transfection over a wider distribution, which may account for the more efficient angiogenesis.

Molecular imaging of endothelial progenitor cell engraftment using contrast-enhanced ultrasound and targeted microbubbles.

AIMS: Imaging methods to track the fate of progenitor cells after their delivery would be useful in assessing the efficacy of cell-based therapies. We hypothesized that contrast-enhanced ultrasound (CEU) using microbubbles targeted to a genetically engineered cell-surface marker on endothelial progenitor cells (EPCs) would allow the targeted imaging of vascular engraftment. METHODS AND RESULTS: Rodent bone marrow-derived EPCs were isolated, cultured, and transfected to express the marker protein, H-2Kk, on the cell surface. Non-transfected EPCs and EPCs transfected with either null plasmid or Firefly luciferase served as controls. Control microbubbles (MB(C)) and microbubbles targeted to H-2Kk expressed on EPCs (MB(H-2Kk)) were constructed. Binding of targeted microbubbles to EPCs was assessed in vitro using a parallel plate flow chamber system. CEU imaging of EPC-targeted microbubbles was assessed in vivo using subcutaneously implanted EPC-supplemented Matrigel plugs in rats. In flow chamber experiments, there was minimal attachment of microbubbles to plated control EPCs. Although numbers of adhered MB(C) were also low, there was greater and more diffuse attachment of MB(H-2Kk) to plated H-2Kk-transfected EPCs. Targeted CEU demonstrated marked contrast enhancement at the periphery of the H-2Kk-transfected EPC-supplemented Matrigel plug for MB(H-2Kk,) whereas contrast enhancement was low for MB(C). Contrast enhancement was also low for both microbubbles within control mock-transfected EPC plugs. The signal intensity within the H-2Kk-transfected EPC plug was significantly greater for MB(H-2Kk) when compared with MB(C). CONCLUSION: Microbubbles targeted to a genetically engineered cell-surface marker on EPCs exhibit specific binding to EPCs in vitro. These targeted microbubbles bind to engrafted EPCs in vivo within Matrigel plugs and can be detected by their enhancement on CEU imaging.

The (Pro)renin receptor: site-specific and functional linkage to the vacuolar H+-ATPase in the kidney.

The (pro)renin receptor ([P]RR) is a transmembrane protein that binds both renin and prorenin with high affinity, increasing the catalytic cleavage of angiotensinogen and signaling intracellularly through mitogen-activated protein kinase activation. Although initially reported as having no homology with any known membrane protein, other studies have suggested that the (P)RR is an accessory protein, named ATP6ap2, that associates with the vacuolar H(+)-ATPase, a key mediator of final urinary acidification. Using in situ hybridization, immunohistochemistry, and electron microscopy, together with serial sections stained with nephron segment-specific markers, we found that (P)RR mRNA and protein were predominantly expressed in collecting ducts and in the distal nephron. Within collecting ducts, the (P)RR was most abundant in microvilli at the apical surface of A-type intercalated cells. Dual-staining immunofluorescence demonstrated colocalization of the (P)RR with the B1/2 subunit of the vacuolar H(+)-ATPase, the ion exchanger that secretes H(+) ions into the urinary space and that associates with an accessory subunit homologous to the (P)RR. In collecting duct/distal tubule lineage Madin-Darby canine kidney cells, extracellular signal-regulated kinase 1/2 phosphorylation, induced by either renin or prorenin, was attenuated by the selective vacuolar H(+)-ATPase inhibitor bafilomycin. The predominant expression of the (P)RR at the apex of acid-secreting cells in the collecting duct, along with its colocalization and homology with an accessory protein of the vacuolar H(+)-ATPase, suggests that the (P)RR may function primarily in distal nephron H(+) transport, recently noted to be, at least in part, an angiotensin II-dependent phenomenon.