Christine Liao

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-ß. 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 106 SCs had no effect, 106 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, 106 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 106 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 µmol/L), urinary protein excretion (9×/÷1 v 64×/÷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/µL, 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).

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.

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.

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.

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.

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.

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.

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.

Impaired recovery of hind limb muscle perfusion following ischemic injury in rodents receiving B vitamin supplementation

Background: B vitamin deficiency has been previously reported to be prevalent in patients with heart failure. However, clinical trials investigating the potential of B vitamin supplementation have failed to show benefit and have suggested some evidence of harm. Negative findings have been hypothesized to be the result of an anti-angiogenic effect associated with B vitamin supplementation. Objective: The current study aimed to determine the impact of vitamin B6 supplementation alone or in combination with folate and B12 on the angiogenic response in a rodent model of hind limb ischemia. Methods: Rats were divided randomly into three groups and fed one of three diets: control diet (CON), high B6 diet (HB6) or high B6+folate+B12 diet (TV). Following five weeks of diet therapy, ischemia was surgically induced in the hind limb. Changes in perfusion and markers of the angiogenic response were studied at either 5 (early) or 10 (late) weeks. Result: Circulating EPCs were reduced in HB6 animals, a finding that reached significance for the TV animals. Quantitative polymerase chain reaction analysis revealed a decline in VEGF and eNOS expression in HB6 and TV groups at week 10. Contrast enhanced ultrasound of the hind limb revealed a significant reduction in perfusion in HB6 and TV animals in comparison to CON at 10 weeks. B vitamin supplementation had no impact on EPC apoptosis or differentiation. Conclusion: Taken together, these results support a negative impact of B vitamin supplementation on the recovery of perfusion following ischemic injury which may be the result of a down-regulation of chemotactic gene expression, leading to lowered EPC recruitment from the bone marrow to the site of ischemic injury. Correspondence to: Mary Keith, Niagara North Family Health Team, 145 Carlton Street Unit #7, St. Catharines, Ontario, Canada; E-mail: mkeith@ niagaranorthfht.ca