Codon-Optimized and de novo-Synthesized E-Selectin/AAV2 Dose-Response Study for Vascular Regeneration Gene Therapy.

OBJECTIVE: This study focuses on dose-response investigation using a codon-optimized and de novo-synthesized E-Selectin/AAV2 (E-Sel/AAV2) vector in preparation for Investigational New Drug (IND)-enabling of subsequent clinical studies. BACKGROUND: Gene therapy is a potential solution for patients suffering from chronic limb-threatening ischemia (CLTI). Understanding the dose for effective gene delivery is crucial for future IND-enabling studies. METHODS: Expression of the codon-optimized E-Selectin gene was assessed by flow cytometry following in vitro cell transfection assay and RT-qPCR for murine limbs injected in vivo with AAV-m-E-Selectin (E-Sel/AAV2). Dose-response studies involved three cohorts of FVB/NJ mice (n=6/group) with escalating log doses of E-Selectin/AAV2 injected intramuscularly (IM) in divided aliquots, ranging from 2×109 VG to 2×1011 VG, into ischemic limbs created by left femoral artery/vein ligation/excision and administration of nitric oxide synthase inhibitor, L-NAME. Limb perfusion, extent of gangrene free limb, functional limb recovery and therapeutic angiogenesis were assessed. RESULTS: Codon-optimized E-Sel/AAV2 gene therapy exhibits superior expression level than WT E-Sel/AAV2 gene therapy both in vitro and in vivo. Mice treated with a high dose (2×1011 VG) of E-Sel/AAV2 showed significantly improved perfusion indices, lower Faber's scores, increased running stamina and neovascularization compared with lower doses tested with control groups, indicating a distinct dose-dependent response. No toxicity was detected in any of the animal groups studied. CONCLUSION: E-Sel/AAV2 Vascular Regeneration Gene Therapy (VRGT) holds promise for enhancing the recovery of ischemic hindlimb perfusion and function, with the effective dose identified in this study as 2×1011 VG aliquots injected IM.

Directing Cell Delivery to Murine Atherosclerotic Aortic Lesions via Targeting Inflamed Circulatory Interface using Nanocarriers.

Stem cell therapy holds significant potential for many inflammatory diseases and regenerative medicine applications. However, delivery of therapeutic cells to specific disease sites after systemic administration without indiscriminate trafficking to other non-target tissues is a major limitation of current cell therapies. Here, we describe a novel nanocarrier-directed targeted cell delivery system that enables cell surface coating with dendrimer nanocarriers containing adhesion moieties to serve as a global positioning system "GPS" to guide circulating cells to targeted lesions and mediate the anchoring of cells at the inflammation site. By exploiting cell surface ligands/receptors selectively and/or molecular moieties that are highly expressed on activated endothelium in pathologic disease states, nanocarrier-coated cells containing the counterpart binding receptors/ligands can be enabled to specifically traffic to and dock at vasculature within target lesions. We demonstrate the efficacy of the I-domain fragment of LFA-1 ( id LFA-1) complexed to modified nanocarriers to facilitate homing of mesenchymal stem cells (MSCs) to inflamed luminal endothelial cells on which ICAM-1 is highly expressed in a murine model of aortic atherosclerosis. Our method can overcome challenges imposed by the high velocity and dynamic circulatory flow of the aorta to successfully deliver MSCs to atherosclerotic regions and allow for docking of the potentially therapeutic and immunomodulating cells. This targeted cell-delivery platform can be tailored for selective systemic delivery of various types of therapeutic cells to different disease areas.

Targeted cell delivery of mesenchymal stem cell therapy for cardiovascular disease applications: a review of preclinical advancements.

Cardiovascular diseases (CVD) continue to be the leading cause of morbidity and mortality globally and claim the lives of over 17 million people annually. Current management of CVD includes risk factor modification and preventative strategies including dietary and lifestyle changes, smoking cessation, medical management of hypertension and cholesterol lipid levels, and even surgical revascularization procedures if needed. Although these strategies have shown therapeutic efficacy in reducing major adverse cardiovascular events such as heart attack, stroke, symptoms of chronic limb-threatening ischemia (CLTI), and major limb amputation significant compliance by patients and caregivers is required and off-target effects from systemic medications can still result in organ dysfunction. Stem cell therapy holds major potential for CVD applications but is limited by the low quantities of cells that are able to traffic to and engraft at diseased tissue sites. New preclinical investigations have been undertaken to modify mesenchymal stem cells (MSCs) to achieve targeted cell delivery after systemic administration. Although previous reviews have focused broadly on the modification of MSCs for numerous local or intracoronary administration strategies, here we review recent preclinical advances related to overcoming challenges imposed by the high velocity and dynamic flow of the circulatory system to specifically deliver MSCs to ischemic cardiac and peripheral tissue sites. Many of these technologies can also be applied for the targeted delivery of other types of therapeutic cells for treating various diseases.

Novel Gene-Modified Mesenchymal Stem Cell Therapy Reverses Impaired Wound Healing in Ischemic Limbs.

OBJECTIVE: Here, we report a new method to increase the therapeutic potential of mesenchymal stem/stromal cells (MSCs) for ischemic wound healing. We tested biological effects of MSCs modified with E-selectin, a cell adhesion molecule capable of inducing postnatal neovascularization, on a translational murine model. BACKGROUND: Tissue loss significantly worsens the risk of extremity amputation for patients with chronic limb-threatening ischemia. MSC-based therapeutics hold major promise for wound healing and therapeutic angiogenesis, but unmodified MSCs demonstrate only modest benefits. METHODS: Bone marrow cells harvested from FVB/ROSA26Sor mTmG donor mice were transduced with E-selectin-green fluorescent protein (GFP)/AAV-DJ or GFP/AAV-DJ (control). Ischemic wounds were created via a 4 mm punch biopsy in the ipsilateral limb after femoral artery ligation in recipient FVB mice and subsequently injected with phosphate-buffered saline or 1×10 6 donor MSC GFP or MSC E-selectin-GFP . Wound closure was monitored daily for 7 postoperative days, and tissues were harvested for molecular and histologic analysis and immunofluorescence. Whole-body DiI perfusion and confocal microscopy were utilized to evaluate wound angiogenesis. RESULTS: Unmodified MSCs do not express E-selectin, and MSC E-selectin-GFP gain stronger MSC phenotype yet maintain trilineage differentiation and colony-forming capability. MSC E-selectin-GFP therapy accelerates wound healing compared with MSC GFP and phosphate-buffered saline treatment. Engrafted MSC E-selectin-GFP manifest stronger survival and viability in wounds at postoperative day 7. Ischemic wounds treated with MSC E-selectin-GFP exhibit more abundant collagen deposition and enhanced angiogenic response. CONCLUSIONS: We establish a novel method to potentiate regenerative and proangiogenic capability of MSCs by modification with E-selectin/adeno-associated virus. This innovative therapy carries the potential as a platform worthy of future clinical studies.

E-Selectin/AAV Gene Therapy Promotes Myogenesis and Skeletal Muscle Recovery in a Mouse Hindlimb Ischemia Model.

The response to ischemia in peripheral artery disease (PAD) depends on compensatory neovascularization and coordination of tissue regeneration. Identifying novel mechanisms regulating these processes is critical to the development of nonsurgical treatments for PAD. E-selectin is an adhesion molecule that mediates cell recruitment during neovascularization. Therapeutic priming of ischemic limb tissues with intramuscular E-selectin gene therapy promotes angiogenesis and reduces tissue loss in a murine hindlimb gangrene model. In this study, we evaluated the effects of E-selectin gene therapy on skeletal muscle recovery, specifically focusing on exercise performance and myofiber regeneration. C57BL/6J mice were treated with intramuscular E-selectin/adeno-associated virus serotype 2/2 gene therapy (E-sel/AAV) or LacZ/AAV2/2 (LacZ/AAV) as control and then subjected to femoral artery coagulation. Recovery of hindlimb perfusion was assessed by laser Doppler perfusion imaging and muscle function by treadmill exhaustion and grip strength testing. After three postoperative weeks, hindlimb muscle was harvested for immunofluorescence analysis. At all postoperative time points, mice treated with E-sel/AAV had improved hindlimb perfusion and exercise capacity. E-sel/AAV gene therapy also increased the coexpression of MyoD and Ki-67 in skeletal muscle progenitors and the proportion of Myh7+ myofibers. Altogether, our findings demonstrate that in addition to improving reperfusion, intramuscular E-sel/AAV gene therapy enhances the regeneration of ischemic skeletal muscle with a corresponding benefit on exercise performance. These results suggest a potential role for E-sel/AAV gene therapy as a nonsurgical adjunct in patients with life-limiting PAD.

Mesenchymal stem cell-based therapy for non-healing wounds due to chronic limb-threatening ischemia: A review of preclinical and clinical studies.

Progressive peripheral arterial disease (PAD) can result in chronic limb-threatening ischemia (CLTI) characterized by clinical complications including rest pain, gangrene and tissue loss. These complications can propagate even more precipitously in the setting of common concomitant diseases in patients with CLTI such as diabetes mellitus (DM). CLTI ulcers are cutaneous, non-healing wounds that persist due to the reduced perfusion and dysfunctional neovascularization associated with severe PAD. Existing therapies for CLTI are primarily limited to anatomic revascularization and medical management of contributing factors such as atherosclerosis and glycemic control. However, many patients fail these treatment strategies and are considered "no-option," thereby requiring extremity amputation, particularly if non-healing wounds become infected or fulminant gangrene develops. Given the high economic burden imposed on patients, decreased quality of life, and poor survival of no-option CLTI patients, regenerative therapies aimed at neovascularization to improve wound healing and limb salvage hold significant promise. Cell-based therapy, specifically utilizing mesenchymal stem/stromal cells (MSCs), is one such regenerative strategy to stimulate therapeutic angiogenesis and tissue regeneration. Although previous reviews have focused primarily on revascularization outcomes after MSC treatments of CLTI with less attention given to their effects on wound healing, here we review advances in pre-clinical and clinical studies related to specific effects of MSC-based therapeutics upon ischemic non-healing wounds associated with CLTI.

Methods and Limitations of Augmenting Mesenchymal Stem Cells for Therapeutic Applications.

Significance: Given their capacity for self-renewal, multilineage differentiation, and immunomodulatory potential, mesenchymal stem cells (MSCs) represent a promising modality of clinical therapy for both regenerative medicine and immune diseases. In this study, we review the key approaches and popular methods utilized to boost potency and modify functions of MSCs for clinical purposes as well as their associated limitations. Recent Advances: Several major domains of cell modification strategies are currently employed by investigators to overcome these deficits and augment the therapeutic potential of MSCs. Priming MSCs with soluble factors or pharmacologic agents as well as manipulating oxygen availability in culture have been demonstrated to be effective biochemical methods to augment MSC potential. Distinct genetic and epigenetic methods have emerged in recent years to modify the genetic expression of target proteins and factors thereby modulating MSCs capacity for differentiation, migration, and proliferation. Physical methods utilizing three-dimensional culture methods and alternative cell delivery systems and scaffolds can be used to recapitulate the native MSC niche and augment their engraftment and viability for in vivo models. Critical Issues: Unmodified MSCs have demonstrated only modest benefits in many preclinical and clinical studies due to issues with cell engraftment, viability, heterogeneity, and immunocompatibility between donor and recipient. Furthermore, unmodified MSCs can have low inherent therapeutic potential for which intensive research over the past few decades has been dedicated to improving cell functionality and potency.

E-Selectin/AAV2/2 Gene Therapy Alters Angiogenesis and Inflammatory Gene Profiles in Mouse Gangrene Model.

For patients with chronic limb-threatening ischemia and limited revascularization options, alternate means for therapeutic angiogenesis and limb salvage are needed. E-selectin is a cell adhesion molecule that is critical for inflammation and neovascularization in areas of wound healing and ischemia. Here, we tested the efficacy of modifying ischemic limb tissue by intramuscular administration of E-selectin/AAV2/2 (adeno-associated virus serotype 2/2) to modulate angiogenic and inflammatory responses in a murine hindlimb gangrene model. Limb appearance, reperfusion, and functional recovery were assessed for 3 weeks after induction of ischemia. Mice receiving E-selectin/AAV2/2 gene therapy had reduced gangrene severity, increased limb and footpad perfusion, enhanced recruitment of endothelial progenitor cells, and improved performance on treadmill testing compared to control group. Histologically, E-selectin/AAV2/2 gene therapy was associated with increased vascularity and preserved myofiber integrity. E-selectin/AAV2/2 gene therapy also upregulated a panel of pro-angiogenic genes yet downregulated another group of genes associated with the inflammatory response. This novel gene therapy did not induce adverse effects on coagulability, or hematologic, hepatic, and renal function. Our findings highlight the potential of E-selectin/AAV2/2 gene therapy for improving limb perfusion and function in patients with chronic limb-threatening ischemia.

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model.

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

Therapeutic angiogenesis in Buerger's disease: reviewing the treatment landscape.

Thromboangiitis obliterans, also known as Buerger's disease, is a rare inflammatory vasculitis that predominantly develops in smokers and characteristically affects the small- and medium-sized peripheral arteries and veins. Patients typically present with extremity claudication, but symptoms may progress to rest pain and tissue loss, especially in those unable to abstain from tobacco use. Unfortunately, traditional medical treatments are largely ineffective and due to the small caliber of affected vessels and lack of suitable distal targets or venous conduits, endovascular and open surgical approaches are often not possible. Eventually, a significant number of patients require major amputation. For these reasons, much research effort has been made in developing techniques of therapeutic angiogenesis to improve limb perfusion, both for atherosclerotic peripheral arterial disease and the smaller subset of patients with critical limb ischemia due to Buerger's disease. Neovascularization in response to ischemia relies on a complex interplay between the local tissue microenvironment and circulating stem and progenitor cells. To date, studies of therapeutic angiogenesis have therefore focused on exploiting known angiogenic factors and stem cells to induce neovascularization in ischemic tissues. This review summarizes the available clinical data regarding the safety and efficacy of various angiogenic therapies, notably injection of naked DNA plasmids, viral gene constructs, and cell-based preparations, and describes techniques for potentiating in vivo efficacy of gene- and cell-based therapies as well as ongoing developments in exosome-based cell-free approaches for therapeutic angiogenesis. Plain Language Title and Summary: A review of available and emerging treatments for improving blood flow and wound healing in patients with Buerger's disease, a rare disorder of blood vessels Buerger's disease is a rare disorder of the small- and medium-sized blood vessels in the arms and legs that almost exclusively develops in young smokers. Buerger's disease causes inflammation in arteries and veins, which leads to blockage of these vessels and reduces blood flow to and from the extremities. Decreased blood flow to the arms and legs can lead to development of nonhealing wounds and infection for which some patients may eventually require amputation. Unfortunately, traditional medical and surgical treatments are not effective in Buerger's disease, so other methods for improving blood flow are needed for these patients. There are several different ways to stimulate new blood vessel formation, both in humans and animal models. The most common treatments involve injection of DNA or viruses that express genes related to blood vessel formation or, alternatively, stem cell-based treatments that help regenerate blood vessels and repair wound tissue. This review explores how safe and effective these various treatments are and describes recent research developments that may lead to better therapies for patients with Buerger's disease and other vascular disorders.

E-Selectin-Overexpressing Mesenchymal Stem Cell Therapy Confers Improved Reperfusion, Repair, and Regeneration in a Murine Critical Limb Ischemia Model.

AIMS: Novel cell-based therapeutic angiogenic treatments for patients with critical limb ischemia may afford limb salvage. Mesenchymal stem cells (MSCs) do not overexpress E-selectin; however, we have previously demonstrated the cell-adhesion molecule's vital role in angiogenesis and wound healing. Thus, we created a viral vector to overexpress E-selectin on MSCs to increase their therapeutic profile. METHODS AND RESULTS: Femoral artery ligation induced hind limb ischemia in mice and intramuscular injections were administered of vehicle or syngeneic donor MSCs, transduced ex vivo with an adeno-associated viral vector to express either GFP+ (MSCGFP) or E-selectin-GFP+ (MSCE-selectin-GFP). Laser Doppler Imaging demonstrated significantly restored reperfusion in MSCE-selectin-GFP-treated mice vs. controls. After 3 weeks, the ischemic limbs in mice treated with MSCE-selectin-GFP had increased footpad blood vessel density, hematoxylin and eosin stain (H&E) ischemic calf muscle sections revealed mitigated muscular atrophy with restored muscle fiber size, and mice were able to run further before exhaustion. PCR array-based gene profiling analysis identified nine upregulated pro-angiogenic/pro-repair genes and downregulated Tumor necrosis factor (TNF) gene in MSCE-selectin-GFP-treated limb tissues, indicating that the therapeutic effect is likely achieved via upregulation of pro-angiogenic cytokines and downregulation of inflammation. CONCLUSION: This innovative cell therapy confers increased limb reperfusion, neovascularization, improved functional recovery, decreased muscle atrophy, and thus offers a potential therapeutic method for future clinical studies.

Rhinella marina oocytes: a suitable alternative expression system for functional characterization of aquaglyceroporins.

Amphibian oocytes have been extensively used for heterologous expression of membrane proteins for studying their biochemical and biophysical properties. So far, Xenopus laevis is the main amphibian used as oocytes source to express aquaglyceroporins in order to assess water and solutes permeability. However, this well-established amphibian model represents a threat to the biodiversity in many countries, especially in those from tropical regions. For that reason, the import of Xenopus laevis is subjected to strict control, which essentially has restricted its use in these regions. Therefore, a wider variety of expression systems for aquaglyceroporins is needed. Rhinella marina is extensively distributed in the Americas and its native range spreads from South America to Texas, US. Here we report the use of Rhinella marina oocytes as an alternative expression system for aquaglyceroporins and demonstrated its suitability to determine the permeability to water and non-ionic solutes. Rhinella marina oocytes were able to functionally express channels from human and the protozoan pathogen Trypanosoma brucei, two very distant organisms on the evolutionary scale. Permeability values obtained from Rhinella marina oocytes expressing members of aquaporin family were similar and comparable to those values reported in the literature for the same channels expressed in Xenopus laevis oocytes.