Anna Bulysheva

Human placenta hydrogel reduces scarring in a rat model of cardiac ischemia and enhances cardiomyocyte and stem cell cultures.

INTRODUCTION Xenogeneic extracellular matrix (ECM) hydrogels have shown promise in remediating cardiac ischemia damage in animal models, yet analogous human ECM hydrogels have not been well development. An original human placenta-derived hydrogel (hpECM) preparation was thus generated for assessment in cardiomyocyte cell culture and therapeutic cardiac injection applications. METHODS AND RESULTS Hybrid orbitrap-quadrupole mass spectrometry and ELISAs showed hpECM to be rich in collagens, basement membrane proteins, and regenerative growth factors (e.g. VEGF-B, HGF). Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes synchronized and electrically coupled on hpECM faster than on conventional cell culture environments, as validated by intracellular calcium measurements. In vivo, injections using biotin-labeled hpECM confirmed its spatially discrete localization to the myocardium proximal to the injection site. hpECM was injected into rat myocardium following an acute myocardium infarction induced by left anterior descending artery ligation. Compared to sham treated animals, which exhibited aberrant electrical activity and larger myocardial scars, hpECM injected rat hearts showed a significant reduction in scar volume along with normal electrical activity of the surviving tissue, as determined by optical mapping. CONCLUSION Placental matrix and growth factors can be extracted as a hydrogel that effectively supports cardiomyocytes in vitro, and in vivo reduces scar formation while maintaining electrophysiological activity when injected into ischemic myocardium. STATEMENT OF SIGNIFICANCE This is the first report of an original extracellular matrix hydrogel preparation isolated from human placentas (hpECM). hpECM is rich in collagens, laminin, fibronectin, glycoproteins, and growth factors, including known pro-regenerative, pro-angiogenic, anti-scarring, anti-inflammatory, and stem cell-recruiting factors. hpECM supports the culture of cardiomyocytes, stem cells and blood vessels assembly from endothelial cells. In a rat model of myocardial infarction, hpECM injections were safely deliverable to the ischemic myocardium. hpECM injections repaired the myocardium, resulting in a significant reduction in infarct size, more viable myocardium, and a normal electrophysiological contraction profile. hpECM thus has potential in therapeutic cardiovascular applications, in cellular therapies (as a delivery vehicle), and is a promising biomaterial for advancing basic cell-based research and regenerative medicine applications.

Gene Electro Transfer of Plasmid Encoding Vascular Endothelial Growth Factor for Enhanced Expression and Perfusion in the Ischemic Swine Heart

Myocardial ischemia can damage heart muscle and reduce the hearts pumping efficiency. This study used an ischemic swine heart model to investigate the potential for gene electro transfer of a plasmid encoding vascular endothelial growth factor for improving perfusion and, thus, for reducing cardiomyopathy following acute coronary syndrome. Plasmid expression was significantly greater in gene electro transfer treated tissue compared to injection of plasmid encoding vascular endothelial growth factor alone. Higher gene expression was also seen in ischemic versus non-ischemic groups with parameters 20 Volts (p<0.03), 40 Volts (p<0.05), and 90 Volts (p<0.05), but not with 60 Volts (p<0.09) while maintaining a pulse width of 20 milliseconds. The group with gene electro transfer of plasmid encoding vascular endothelial growth factor had increased perfusion in the area at risk compared to control groups. Troponin and creatine kinase increased across all groups, suggesting equivalent ischemia in all groups prior to treatment. Echocardiography was used to assess ejection fraction, cardiac output, stroke volume, left ventricular end diastolic volume, and left ventricular end systolic volume. No statistically significant differences in these parameters were detected during a 2-week time period. However, directional trends of these variables were interesting and offer valuable information about the feasibility of gene electro transfer of vascular endothelial growth factor in the ischemic heart. The results demonstrate that gene electro transfer can be applied safely and can increase perfusion in an ischemic area. Additional study is needed to evaluate potential efficacy.

Vascular endothelial growth factor-A gene electrotransfer promotes angiogenesis in a porcine model of cardiac ischemia

This study aimed to assess safety and therapeutic potential of gene electrotransfer (GET) as a method for delivery of plasmid encoding vascular endothelial growth factor A (VEGF-A) to ischemic myocardium in a porcine model. Myocardial ischemia was induced by surgically occluding the left anterior descending coronary artery in swine. GET following plasmid encoding VEGF-A injection was performed at four sites in the ischemic region. Control groups either received injections of the plasmid without electrotransfer or injections of the saline vehicle. Animals were monitored for 7 weeks and the hearts were evaluated for angiogenesis, myocardial infarct size and left ventricular contractility. Arteriograms suggest growth of new arteries as early as 2 weeks after treatment in electrotransfer animals. There is a significant reduction of infarct area and left ventricular contractility is improved in GET-treated group compared with controls. There was no significant difference in mortality of animals treated with GET of plasmid encoding VEGF-A from the control groups. Gene delivery of plasmid encoding VEGF-A to ischemic myocardium in a porcine model can be accomplished safely with potential for myocardial repair and regeneration.

Recellularized human dermis for testing gene electrotransfer ex vivo.

Gene electrotransfer (GET) is a proven and valuable tool for in vivo gene delivery to a variety of tissues such as skin, cardiac muscle, skeletal muscle, and tumors, with controllable gene delivery and expression levels. Optimizing gene expression is a challenging hurdle in preclinical studies, particularly for skin indications, due to differences in electrical conductivity of animal compared to human dermis. Therefore, the goal of this study was to develop an ex vivo model for GET using recellularized human dermis to more closely mimic human skin. Decellularized human dermis (DermACELL(®)) was cultured with human dermal fibroblasts and keratinocytes for 4 weeks. After one week of fibroblast culture, fibroblasts infiltrated and dispersed throughout the dermis. Air-liquid interface culture led to epithelial cell proliferation, stratification and terminal differentiation with distinct basal, spinous, granular and cornified strata. Firefly luciferase expression kinetics were evaluated after GET of recellularized constructs for testing gene delivery parameters to skin in vitro. Elevated luciferase expression persisted up to a week following GET compared to controls without electrotransfer. In summary, recellularized dermis structurally and functionally resembled native human skin in tissue histological organization and homeostasis, proving an effective 3D human skin model for preclinical gene delivery studies.

Plasma-activated air mediates plasmid DNA delivery in vivo

Plasma-activated air (PAA) provides a noncontact DNA transfer platform. In the current study, PAA was used for the delivery of plasmid DNA in a 3D human skin model, as well as in vivo. Delivery of plasmid DNA encoding luciferase to recellularized dermal constructs was enhanced, resulting in a fourfold increase in luciferase expression over 120 hours compared to injection only (P < 0.05). Delivery of plasmid DNA encoding green fluorescent protein (GFP) was confirmed in the epidermal layers of the construct. In vivo experiments were performed in BALB/c mice, with skin as the delivery target. PAA exposure significantly enhanced luciferase expression levels 460-fold in exposed sites compared to levels obtained from the injection of plasmid DNA alone (P < 0.001). Expression levels were enhanced when the plasma reactor was positioned more distant from the injection site. Delivery of plasmid DNA encoding GFP to mouse skin was confirmed by immunostaining, where a 3-minute exposure at a 10 mm distance displayed delivery distribution deep within the dermal layers compared to an exposure at 3 mm where GFP expression was localized within the epidermis. Our findings suggest PAA-mediated delivery warrants further exploration as an alternative approach for DNA transfer for skin targets.

Thermal Assisted In Vivo Gene Electrotransfer

Gene electrotransfer is an effective approach for delivering plasmid DNA to a variety of tissues. Delivery of molecules with electric pulses requires control of the electrical parameters to achieve effective delivery. Since discomfort or tissue damage may occur with high applied voltage, the reduction of the applied voltage while achieving the desired expression may be an important improvement. One possible approach is to combine electrotransfer with exogenously applied heat. Previous work performed in vitro demonstrated that increasing temperature before pulsing can enhance gene expression and made it possible to reduce electric fields while maintaining expression levels. In the study reported here, this combination was evaluated in vivo using a novel electrode device designed with an inserted laser for application of heat. The results obtained in this study demonstrated that increased temperature during electrotransfer increased expression or maintained expression with a reduction in applied voltage. With further optimization this approach may provide the basis for both a novel method and a novel instrument that may greatly enhance translation of gene electrotransfer.

Coalesced thermal and electrotransfer mediated delivery of plasmid DNA to the skin

Efficient gene delivery and expression in the skin can be a promising minimally invasive technique for therapeutic clinical applications for immunotherapy, vaccinations, wound healing, cancer, and peripheral artery disease. One of the challenges for efficient gene electrotransfer (GET) to skin in vivo is confinement of expression to the epithelium. Another challenge involves tissue damage. Optimizing gene expression profiles, while minimizing tissue damage are necessary for therapeutic applications. Previously, we established that heating pretreatment to 43 °C enhances GET in vitro. We observed a similar trend in vivo, with an IR-pretreatment for skin heating prior to GET. Currently, we tested a range of GET conditions in vivo in guinea pigs with and without preheating the skin to ~43 °C. IR-laser heating and conduction heating were tested in conjunction with GET. In vivo electrotransfer to the skin by moderately elevating tissue temperature can lead to enhanced gene expression, as well as achieve gene transfer in epidermal, dermal, hypodermal and muscle tissue layers.

Direct Crystal Formation from Micronized Bone and Lactic Acid: The Writing on the Wall for Calcium-Containing Crystal Pathogenesis in Osteoarthritis?"

Introduction Pathological calcium-containing crystals accumulating in the joints, synovial fluid, and soft tissues are noted in most elderly patients, yet arthritic crystal formation remains idiopathic. Interestingly, elevated lactic acid and bone erosion are frequently among the comorbidities and clinical features of patients with highest incidence of crystal arthropathies. This work shows that bone particulates (modeling bone erosion) dissolve in lactic acid and directly generate crystals, possibly presenting a mechanism for crystal accumulation in osteoarthritis. Methods and Results Micronized human bone (average particle size of 160μm x 79μm) completely dissolved in lactic acid in 48 hours, and in synovial fluid with 500mM lactic acid in 5 days, generating birefringent rhomboid and rod-shaped crystals. SEM analysis with energy dispersive x-ray spectroscopy of these crystals showed average dimensions of around 2μm x 40μm, which contained oxygen, calcium and phosphorous at 8.64:1.85:1. Raman spectroscopy of the generated crystals further showed 910/cm and 1049/cm peaks, aligning with calcium oxalate monohydrate and calcium pyrophosphate, respectively. Conclusions This work shows that lactic acid and micronized mineralized bone together directly generate calcium-containing crystals. These observations may provide insights into the elusive etiology of arthritis with crystal involvement, possibly indicating lactic acid as a clinical target for treatment.

VEGF-B electrotransfer mediated gene therapy induces cardiomyogenesis in a rat model of cardiac ischemia

Atherosclerosis induced myocardial infarction (MI) continues to be a major public health concern. Regenerative therapies that restore cardiac muscle cells are largely absent. The rate of cardiomyogenesis in adults is insufficient to compensate for MI damage. In this study, we explored the capacity of a gene therapy approach to promote cardiomyogenesis. We hypothesized that VEGF-B, critical during fetal heart development, could promote cardiomyogenesis in adult ischemic hearts. Gene electrotransfer (GET), a physical method of in vivo gene delivery, was adapted to the rat model of MI. Favorable pulsing parameters were then used for delivery of pVEGF-B and compared to a sham control in terms of infarct size, cardiomyocyte proliferation and presence of new cardiomyocytes. Ki67 immunoreactivity was used for proliferation analysis. Newly synthetized DNA was labeled with BrdU to identify new cells post-infarction. Cardiac troponin co-localization indicated proliferating and new cardiomyocytes histologically. Eight weeks post-treatment, GET pVEGF-B treated hearts had significantly smaller infarcts than the sham control group (p < 0.04). Proliferating and new cardiomyocytes were only present in the GET of pVEGF-B group, and absent in the controls. In summary, GET pVEGF-B promoted cardiomyogenesis post-MI, demonstrating for the first time direct evidence of myocardial regeneration post-infarction.

Gene Electro Transfer to Left Ventricular Myocardium in Rat and Porcine Models

Gene electro transfer to the left ventricular myocardium is a promising technique for delivery of therapeutic genes for treatment of ischemia, myocardial infarction and heart failure directly to the effected myocardium. There are multiple variables to consider for efficient gene delivery and gene expression modulation, including electro transfer parameters, timing and duration of pulses relative to the electrocardiogram and ischemic state. Here we report establishing a small animal model and a large animal model for gene delivery to non-ischemic and ischemic left ventricular myocardium. Gene expression was evaluated histologically for location of expression within the myocardium as well as quantitatively via ELISA. We developed electro transfer electrodes and protocols that are safe and result robust gene expression. These animal models allow for evaluation of therapeutic potential of particular gene delivery as well as translation to clinical settings.