Therapy For Critical Limb Ischemia Research Articles

A micro-fragmented collagen gel as a cell-assembling platform for critical limb ischemia repair

Critical limb ischemia (CLI) is a devastating disease characterized by the progressive blockage of blood vessels. Although the paracrine effect of growth factors in stem cell therapy made it a promising angiogenic therapy for CLI, poor cell survival in the harsh ischemic microenvironment limited its efficacy. Thus, an imperative need exists for a stem-cell delivery method that enhances cell survival. Here, a collagen microgel (CMG) cell-delivery scaffold (40 × 20 μm) was fabricated via micro-fragmentation from collagen–hyaluronic acid polyionic complex to improve transplantation efficiency. Culturing human adipose-derived stem cells (hASCs) with CMG enabled integrin receptors to interact with CMG to form injectable 3-dimensional constructs (CMG-hASCs) with a microporous microarchitecture and enhanced mass transfer. CMG-hASCs exhibited higher cell survival (p < 0.0001) and angiogenic potential in tube formation and aortic ring angiogenesis assays than cell aggregates. Injection of CMG-hASCs intramuscularly into CLI mice increased blood perfusion and limb salvage ratios by 40 % and 60 %, respectively, compared to cell aggregate-treated mice. Further immunofluorescent analysis revealed that transplanted CMG-hASCs have greater muscle regenerative and angiogenic potential, with enhanced cell survival than cell aggregates (p < 0.05). Collectively, we propose CMG as a cell-assembling platform and CMG-hASCs as promising therapeutics to treat CLI.

Stem Cell Therapy in Critical Limb Ischemia.

Critical limb ischemia (CLI), a serious outcome of peripheral artery disease, is frequently associated with morbid outcomes. The available treatment modalities do not provide satisfactory results, leading to marked morbidities such as joint contracture and amputations, resulting in a high economic burden. The peripheral vascular disease tends to cause more morbidity in patients with diabetes and atherosclerosis, given the pre-existing compromised perfusion of medium and small vessels in diabetic patients. With surgical procedures, the chance of vascular compromise further increases, inducing a significantly greater rate of amputation. Hence, the need for nonsurgical treatment modalities such as stem cell therapy (SCT), which promotes angiogenesis, is warranted. In CLI, SCT acts through neovascularization and the development of collateral arteries, which increases blood supply to the soft tissues of the ischemic limb, providing satisfactory outcomes. An electronic database search was performed in PubMed, SCOPUS, EMBASE, and ScienceDirect to identify published clinical trial data, research studies, and review articles on stem cell therapy in critical limb ischemia. The search resulted in a total of 2391 results. Duplicate articles screening resulted in 565 articles. In-depth screening of abstracts and research titles excluded 520 articles, yielding 45 articles suitable for full-text review. On review of full text, articles with overlapping and similar results were filtered, ending in 25 articles. SCT promotes arteriogenesis, and bone marrow-derived mesenchymal stromal cells produce significant effects like reduced morbidity, improved amputation-free survival (AFS ) rate, and improved distal perfusion even in "no-option" CLI patients. SCT is a promising treatment modality for CLI patients, even in those in whom endovascular and revascularization procedures are impossible. SCT assures a prolonged AFS rate, improved distal perfusion, improved walking distances, reduced amputation rates, and increased survival ratio, and is well-tolerated.

Intraprocedural, Intra-Arterial CT Foot Perfusion Examination for Assessment of Endovascular Therapy in Patients With Critical Limb Ischemia: A Prospective Pilot Study.

Current techniques to evaluate computed tomography (CT) foot perfusion in patients with critical limb ischemia use high contrast doses and cannot be used during endovascular procedures. CT perfusion of the foot with intra-arterial contrast injection during endovascular treatment in a hybrid angiography CT suite might solve these problems. The main objective of this study was to evaluate whether intra-arterial CT foot perfusion using a hybrid CT angiosystem is feasible during endovascular treatment for critical limb ischemia. This prospective pilot study investigated intraprocedural, intra-arterial CT perfusion of the foot using a hybrid CT angiosystem in 12 patients before and after endovascular treatment for critical limb ischemia. Time to peak (TTP) and arterial blood flow were measured before and after treatment and compared using a paired t test. All 24 CT perfusion maps could be calculated adequately. The contrast volume used for one perfusion CT scan was 4.8 ml. The mean TTP before treatment was 12.8 seconds (standard deviation [SD] 2.8) and the mean TTP posttreatment was 8.4 seconds (SD 1.7), this difference being statistically significant (p=.001). Tendency toward increased blood flow after treatment, 340 ml/min/100 ml (SD 174) vs 514 ml/min/100 ml (SD 366) was noticed (p=.104). The mean effective radiation dose was 0.145 mSv per scan. Computed tomography perfusion of the foot with low contrast dose intra-arterial contrast injection during endovascular treatment in a hybrid angiography CT suite is a feasible technique. Intra-arterial CT foot perfusion using a hybrid CT-angiography system is a feasible new technique during endovascular therapy for critical limb ischemia to assess the results of the treament. Future research is necessary in defining endpoints of endovascular treatment and establishing its role in limb salvage prognostication.

Transplantation of adipose tissue-derived microvascular fragments promotes therapy of critical limb ischemia

BackgroundAdipose tissue-derived microvascular fragments are functional vessel segments derived from arterioles, capillaries, and veins. Microvascular fragments can be used as vascularization units in regenerative medicine and tissue engineering containing microvascular networks. However, the in vivo therapeutic and vascularization properties of human microvascular fragments have not been investigated.MethodsIn this study, we isolated microvascular fragments, stromal vascular fractions, and mesenchymal stem cells from human lipoaspirate and studied their therapeutic efficacy and in vivo vasculogenic activity in a murine model of hindlimb ischemia. In addition, in vivo angiogenic activity and engraftment of microvascular fragments into blood vessels were measured using Matrigel plug assay.ResultsBoth microvascular fragments and stromal vascular fractions contain not only mesenchymal stem cells but also endothelial progenitor cells. In a Matrigel plug assay, microvascular fragments increased the number of blood vessels containing red blood cells more than mesenchymal stem cells and stromal vascular fractions did. The engraftment of the microvascular fragments transplanted in blood vessels within the Matrigel plug significantly increased compared to the engraftment of mesenchymal stem cells and stromal vascular fractions. Moreover, intramuscular injection of microvascular fragments markedly increased blood flow in the ischemic hindlimbs and alleviated tissue necrosis compared to that of mesenchymal stem cells or stromal vascular fractions. Furthermore, transplanted microvascular fragments formed new blood vessels in ischemic limbs.ConclusionsThese results suggest that microvascular fragments show improved engraftment efficiency and vasculogenic activity in vivo and are highly useful for treating ischemic diseases and in tissue engineering.Graphical Adipose tissue-derived microvascular fragments are vascularization units in regenerative medicine and tissue engineering containing microvascular networks. Intramuscular injection of microvascular fragments markedly increased blood flow in the ischemic hindlimbs and alleviated tissue necrosis. The present study suggests that microvascular fragments show improved engraftment efficiency and vasculogenic activity in vivo and are highly useful for treating ischemic diseases and in tissue engineering.

Life Satisfaction in Patients with Critical Limb Ischemia Following Vascular Surgery and Endovascular Therapy

Objective: The purpose of this research is to assess the efficacy of vascular surgery and endovascular therapy for critical limb ischemia. Study Design: Prospective study Place and Duration: This prospective study was conducted at Dow International Medical College and Dow University of Health Sciences, OJHA Campus, Karachi in the period from April, 2022 to September, 2022. Methods: Total 90 patients had critical limb ischemia were presented. All the patients were enrolled after taking informed written consent. Included patients were admitted for revascularization and divided equally in two groups. Group I received vascular surgery in 45 cases and 45 cases of group II received endovascular treatment (PTA). At 3 months post-revascularization, all patients were evaluated using the ankle-brachial index (ABI), the 6-minute walking test, and the Nottingham Health Profile (NHP) questionnaire. SPSS 22.0 was used to analyze all data. Results: The mean age of the included cases in group I was 64.6±5.57 years and in group II mean age was 69.5±8.44 years. There were majority males 33 (73.3%) in group I and 36 (80%) in group II. Comorbidities were DM, HTN, Hyperlipidemia and CAD among all cases. ABI and 6-minute walking distance improved significantly three months after the treatment in both groups. At six months post-procedure, both groups had similar NHP Part I and Part II total scores. However, the surgical revascularization group had substantially higher domain scores in social isolation and physical abilities with p value &lt;0.012. Conclusion: Our findings indicate that patients undergoing surgical revascularization or PTA for SFA stenosis have comparable overall scores on the NHP questionnaire three months after revascularization. Subjects who undergo surgical revascularization score significantly higher than those who undergo percutaneous transluminal angioplasty (PTA) on the NHP's social isolation and physical ability dimensions. Keywords: Peripheral bypass surgery, Peripheral arterial disease, Quality of life, Endovascular treatment

CO delivery nanosystem based on regenerative bioactive zinc MOFs highlights intercellular crosstalk for enhanced vascular remodeling in CLI therapy

Therapeutic angiogenesis is a promising approach for the treatment of critical limb ischemia (CLI), but always face failure in clinical trials. The existing strategies primarily regulate a population of independent cells and ignore intercellular crosstalk, which may constrain efficacy. Inspired by the “self-healing” of organisms, we proposed a novel concept of “biomimetic dual cellular co-regulation”, i.e., regulating both vascular endothelial cells (VECs) or macrophages (Mφs), the protagonists in ischemic repair, and highlighting intercellular crosstalk to break the efficiency bottleneck of conventional strategies. Herein, bioactive zinc metal–organic frameworks (Zn-MOFs)-based carbon monoxide (CO) delivery nanoparticles coated with hyaluronic acid (HA)-CAG peptide (denoted as HA-CAG@ZIF8@CO) were developed, which can simultaneously target and regulate VECs and Mφs and enhance their interactions, with a view to amplify the synergistic cooperation in vascular remodeling. HA-CAG@ZIF8@CO promoted VECs morphogenesis through zinc ion and CO release, normalized microenvironment, and induced Mφ polarization to pro-healing M2-type, which further amplified angiogenesis through the secretion of pro-angiogenic cytokines. Excitingly, the intrinsic communication between VECs and M2-type Mφs enabled the significant enhancement of angiogenic activity. In summary, the dual cellular co-regulatory nanomedicine HA-CAG@ZIF8@CO modulates the intercellular crosstalk and represents a new possibility for therapeutic angiogenesis with clinical implications for ischemic repair.

Uniformly Sized Stem Cell Spheroids for Treatment of Hind Limb Ischemia: Size Effect

AbstractSpheroid‐based cell delivery has been demonstrated as an effective strategy in stem cell therapy of critical limb ischemia. However, massive generation of uniform‐sized spheroids still remains a big challenge. In addition, how spheroid size influences its therapeutic efficacy remains an open question. Herein, patterned poly(2‐hydroxyethyl methacrylate) hydrogel films synthesized in situ in cell culture plates are used to generate adipose‐derived stromal cell (ADSC) spheroids. This method not only allows for the production of uniform‐sized spheroids but also large‐scale production (≈60 000 spheroids per plate). Using this method, monodisperse ADSC spheroids with three different sizes are generated to study spheroid size effect in ischemia treatment. It is found that different size spheroids exhibit different behaviors in extracellular matrix (ECM) expression and paracrine secretion. Larger spheroids express more ECM proteins. Paracrine secretion of hypoxia‐induced survival factors, anti‐apoptotic and angiogenic factors is enhanced to a larger degree in larger spheroids. Finally, using a mouse model of hind limb ischemia, it is demonstrated that the therapeutic efficacy of the spheroids increases with increasing spheroid size. The improved therapeutic efficacy is attributed to enhanced survival and paracrine secretion of larger spheroids, which is attributed to the higher degree of hypoxic environment of these spheroids.

Surgical and cell therapy in critical limb ischemia: Current evidence and rationale for combined treatment with special focus on diabetic patients

Critical limb ischemia (CLI) is considered the end-stage of peripheral arterial disease, with a prevalence between 2% and 4% in the general population and more than 15% in older adults. One-year major amputation rate can reach 30%, and diabetic patients are five times more likely to develop CLI than nondiabetics. The vascular damage and the complexity in the anatomical extension of the lesions are also worse in people with diabetes with poorer outcomes after vascularization attempts. Following the classifications suggested by international guidelines, we can define the presence of CLI and have a precise evaluation of the amputation risk and the best revascularization procedure for the patient. Nowadays, new endovascular techniques and devices make it possible to treat tibial vessels and even arteries below the ankle with promising initial results. Nevertheless, the re-occlusions rate and the need to re-do treatments at 1 year remain between 30% and 50%. The disease progression and hyperplasia can because it. However, the damage at the microcirculatory level can also lead to a decrease in tissue runoff and an increase in peripheral resistance, which determine the revascularization failure. In the last 20 years, several trials have been designed to avoid amputation in patients with no surgical options. The aim is to find a valid cellular base therapy to create a new vessel web in the ischemic tissue based on the angiogenetic power that stem cells have already demonstrated in vitro and animal studies. Different types of cells have been tested with different concentrations and administration routes with promising results. CD34+ Mononuclear cells, Mesenchymal stem cells, growth factors have demonstrated their contribution to the neo-angiogenesis in ischemic areas. At Abu Dhabi Stem Cells Center, we created a cellular cocktail as an adjunct treatment to surgical revascularization. We think that acting at the microcirculatory and immunological level. We may reduce postsurgery hyperplasia and increase tissue perfusion, ultimately prolonging the patency of revascularization procedures.

ROS-scavenging hybrid hydrogel for genetically engineered stem cell delivery and limb ischemia therapy

Stem cell therapy is a promising strategy for the treatment of ischemic diseases such as critical limb ischemia (CLI). The major obstacle is the poor survival rate and low efficiency of the implanted stem cells. Biomimetic scaffolds such as injectable hydrogels have shown the ability to promote cell retention and survival. However, the local ischemic microenvironment especially the high level of reactive oxygen species (ROS) usually induces cell loss and dysfunction, thereby leading to unsatisfactory therapeutic outcomes. Herein, we developed a new injectable hybrid hydrogel composed of natural type I collagen and melanin nanoparticles (MeNPs) for the delivery of Gremlin1 (Grem1)-overexpressed mesenchymal stem cells (Grem1-MSCs) and CLI therapy. After local injection of this cell delivery system into the ischemic limbs of mice, the thermal-triggered in situ gelation of collagen could maintain Grem1-MSCs at the injection site and MeNPs entrapped in the hydrogel framework could modulate the ischemic microenvironment via scavenging ROS to promote the survival of implanted stem cells. With this improved cell retention and survival, Grem1-MSCs could constantly secrete high concentration of Grem1, a newly discovered proangiogenic factor that can promote angiogenesis by activating the phosphoinositide 3-kinase (PI3K)-AKT signaling pathway, thereby leading to improved blood perfusion in the ischemic limbs and superior limb salvage. The strategy developed herein offers a promising therapeutic option for the effective cell-based treatment of ischemic diseases.

Deciphering the in vivo Dynamic Proteomics of Mesenchymal Stem Cells in Critical Limb Ischemia.

ObjectiveRegenerative therapy using mesenchymal stem cells (MSC) is a promising therapeutic method for critical limb ischemia (CLI). To understand how the cells are involved in the regenerative process of limb ischemia locally, we proposed a metabolic protein labeling method to label cell proteomes in situ and then decipher the proteome dynamics of MSCs in ischemic hind limb.Methods and ResultsIn this study, we overexpressed mutant methionyl-tRNA synthetase (MetRS), which could utilize azidonorleucine (ANL) instead of methionine (Met) during protein synthesis in MSCs. Fluorescent non-canonical amino-acid tagging (FUNCAT) was performed to detect the utilization of ANL in mutant MSCs. Mice with hindlimb ischemia (HLI) or Sham surgery were treated with MetRSmut MSCs or PBS, followed by i.p. administration of ANL at days 0, 2 6, and 13 after surgery. FUNCAT was also performed in hindlimb tissue sections to demonstrate the incorporation of ANL in transplanted cells in situ. At days 1, 3, 7, and 14 after the surgery, laser doppler imaging were performed to detect the blood reperfusion of ischemic limbs. Ischemic tissues were also collected at these four time points for histological analysis including HE staining and vessel staining, and processed for click reaction based protein enrichment followed by mass spectrometry and bioinformatics analysis. The MetRSmut MSCs showed strong green signal in cell culture and in HLI muscles as well, indicating efficient incorporation of ANL in nascent protein synthesis. By 14 days post-treatment, MSCs significantly increased blood reperfusion and vessel density, while reducing inflammation in HLI model compared to PBS. Proteins enriched by click reaction were distinctive in the HLI group vs. the Sham group. 34, 31, 49, and 26 proteins were significantly up-regulated whereas 28, 32, 62, and 27 proteins were significantly down-regulated in HLI vs. Sham at days 1, 3, 7, and 14, respectively. The differentially expressed proteins were more pronounced in the pathways of apoptosis and energy metabolism.ConclusionIn conclusion, mutant MetRS allows efficient and specific identification of dynamic cell proteomics in situ, which reflect the functions and adaptive changes of MSCs that may be leveraged to understand and improve stem cell therapy in critical limb ischemia.

Comparison of sympathectomy and cilostazol treatment results in non-revascularized critical leg ischemia

ABSTRACT&#x0D; Objective: The aim of this retrospective study is to compare the efficacy of sympathectomy and cilostazol therapy in critical limb ischemia that cannot be revascularized.&#x0D; Material and Method: This study was retrospectively conducted on 30 patients who underwent lumbar sympathectomy (Group 1) and received cilostazol treatment (Group 2) between January 2017 and August 2020. Demographic data, comorbidity, complications, wound healing, walking distance, and pain scale records of the patients were determined by examining the hospital registry system and statistical analysis was performed.&#x0D; Results: In the study, no significant difference was found between the two groups in terms of walking distance and ischemic pain in the statistical analysis of the data before treatment, at the 3rd, 6th, 12th, and 24th months (p&gt; 0.05). However, a statistically significant difference was found between the pre-treatment data and the data at the 3rd, 6th, 12th, and 24th months in both Group 1 and Group 2 in terms of walking distance and ischemic pain (p˂0.001 ). In the time periods followed in both groups, it was observed that there was an increase in walking distance and a decrease in ischemic pain.&#x0D; Conclusion: Cilostazol treatment may be preferred as a good alternative treatment method compared to lumbar sympathectomy in critical leg ischemia.&#x0D; Keywords: Critical leg ischemia; sympathectomy; cilostazol

Decellularized adipose tissue scaffolds guide hematopoietic differentiation and stimulate vascular regeneration in a hindlimb ischemia model

Cellular therapies to stimulate therapeutic angiogenesis in individuals with critical limb ischemia (CLI) remain under intense investigation. In this context, the efficacy of cell therapy is dependent on the survival, biodistribution, and pro-angiogenic paracrine signaling of the cells transplanted. Hematopoietic progenitor cells (HPC) purified from human umbilical cord blood using high aldehyde dehydrogenase-activity (ALDHhi cells) and expanded ex vivo, represent a heterogeneous mixture of progenitor cells previously shown to support limb revascularization in mouse models of CLI. The objectives of this study were to investigate the utility of bioscaffolds derived from human decellularized adipose tissue (DAT) to guide the differentiation of seeded HPC in vitro and harness the pro-angiogenic capacity of HPC at the site of ischemia after implantation in vivo. Probing whether the DAT scaffolds altered HPC differentiation, label-free quantitative mass spectrometry and flow cytometric phenotype analyses indicated that culturing the HPC on the DAT scaffolds supported their differentiation towards the pro-angiogenic monocyte/macrophage lineage at the expense of megakaryopoiesis. Moreover, implantation of HPC in DAT scaffolds within a unilateral hindlimb ischemia model in NOD/SCID mice increased cell retention at the site of ischemia relative to intramuscular injection, and accelerated the recovery of limb perfusion, improved functional limb use and augmented CD31+ capillary density when compared to DAT implantation alone or saline-injected controls. Collectively, these data indicate that cell-instructive DAT scaffolds can direct therapeutic HPC differentiation towards the monocyte/macrophage lineage and represent a promising delivery platform for improving the efficacy of cell therapies for CLI.

Xenotransplantation of neonatal porcine bone marrow-derived mesenchymal stem cells improves murine hind limb ischemia through lymphangiogenesis and angiogenesis.

The clinical utility of stem cell therapy for peripheral artery disease has not been fully discussed, and one obstacle is limited donor supplies. In this study, we attempted to rescue mouse ischemic hind limb by xenotransplantation of neonatal porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs). Neonatal porcine bone marrow-derived mesenchymal stem cells were transplanted to ischemic hind limbs of male C57BL/6J mice (npBM-MSCs group). Mice with syngeneic transplantation of mouse BM-MSCs (mBM-MSCs group) were also prepared for comparison. The angiogenic effects were evaluated by recovery of blood flow on laser Doppler imaging, histologic findings, and genetic and protein levels of angiogenic factors. Regarding laser Doppler assessments, blood flow in the hind limb was rapidly recovered in the npBM-MSCs group, compared with that in the mBM-MSCs group (P=.016). Compared with the mBM-MSCs group, the npBM-MSCs group had early and prominent lymphangiogenesis [P<.05 on both post-operative days (PODs) 3 and 7] but had similar angiogenesis. Regarding genomic assessments, xenotransplantation of npBM-MSCs enhanced the expressions of both porcine and murine Vegfc in the hind limbs by POD 3. Interestingly, the level of murine Vegfc expression was significantly higher in the npBM-MSCs group than in the mBM-MSCs group on PODs 3 and 7 (P<.001 for both). Furthermore, the secreted VEGFC protein level was higher from npBM-MSCs than from mBM-MSCs (P<.001). Xenotransplantation of npBM-MSCs contributed to the improvement of hind limb ischemia by both angiogenesis and lymphangiogenesis, especially promotion of the latter. npBM-MSCs may provide an alternative to autologous and allogeneic MSCs for stem cell therapy of critical limb ischemia.

Main Factors Predicting Nonresponders to Autologous Cell Therapy for Critical Limb Ischemia in Patients With Diabetic Foot.

Autologous cell therapy (ACT) is a new treatment for patients with no-option critical limb ischemia (NO-CLI). We evaluated the factors involved in the nonresponse to ACT in patients with CLI and diabetic foot. Diabetic patients (n = 72) with NO-CLI treated using ACT in our foot clinic over a period of 8 years were divided into responders (n = 57) and nonresponders (n = 15). Nonresponder was defined as an insufficient increase in transcutaneous oxygen pressure by <5 mm Hg, 3 months after ACT. Patient demographics, diabetes duration and treatment, and comorbidities as well as a cellular response to ACT, limb-related factors, and the presence of inherited thrombotic disorders were compared between the 2 groups. The main independent predictors for an impaired response to ACT were heterozygote Leiden mutation (OR 10.5; 95% CI, 1.72-4) and homozygote methylenetetrahydrofolate reductase (MTHFR 677) mutation (OR 3.36; 95% CI, 1.0-14.3) in stepwise logistic regression. Univariate analysis showed that lower mean protein C levels (P = .041) were present in nonresponders compared with responders. In conclusion, the significant predictors of an impaired response to ACT in diabetic patients with NO-CLI were inherited thrombotic disorders.

The current state of endovascular intervention for critical limb ischemia: A systematic review

The treatment of critical limb ischemia (CLI) has long been a “hot spot” in medical science. It is widely believed that revascularization is the cornerstone of CLI therapy. However, there is currently no consensus on the best revascularization approach. Traditional open surgery is traumatic and associated with many complications. In recent years, great progress has been witnessed in terms of endovascular technology, gradually replacing open surgery in the treatment of CLI. In this review, the role of endovascular therapies in clinical practice, including conventional percutaneous transluminal angioplasty, bare-metal stent, and innovated drug-coated balloon, drug-eluting stent, bioresorbable vascular scaffold, cutting balloon angioplasty, atherectomy, intravascular lithotripsy, cryoplasty, and percutaneous deep venous arterialization is discussed.

Mechanisms of ischemic skeletal muscle regeneration mediated by mechanically constrained human allogeneic mesenchymal stromal cells

Background and Hypothesis: Critical limb threatening ischemia (CLTI) is the end stage of peripheral arterial disease (PAD). CLTI presents a significant risk for lower extremity amputation, especially in diabetic patients with poor options for revascularization. Additionally, using a novel diabetic mouse model of CLTI, we have shown that IM injection of 3D cultured MSCs (spheroids) is more effective in promoting skeletal muscle regeneration of ischemic muscle than monolayer cultured MSC. We hypothesize that this result is due to the mechanical constrained nature of the MSC in spheroid form, which has been shown to alter the cellular phenotype. Alginate encapsulated cells are another form of mechanical confinement, and have additional benefits including resistance to immunological attack, making them a practical therapy for treatment of CLTI patients. &#x0D; &#x0D; Project Methods: In this work, cells were encapsulated in 2% alginate using a centrifugation method. Media from these cells along with others were analyzed for IL-10 and IL-33 using ELISA. Mouse tissue samples were stained with WGA-555 antibody and analyzed using a scanning microscope. Effects on tissue perfusion were measured with Laser Doppler Perfusion Imaging. At the time of this abstract, the media from encapsulated and naked MSCs is being used to culture myoblasts, looking at cell growth. &#x0D; &#x0D; Results: ELISA results did not show significant increases in IL-10 or IL-33 for encapsulated vs. non-encapsulated cells. LDPI has shown an increased perfusion rate for hindlimbs treated with encapsulated MSCs vs naked MSCs. Muscle fiber analysis is ongoing, but initial data appears promising. &#x0D; &#x0D; Conclusion and Potential Impact: This experiment provides a starting point for improving and expanding cell therapy for critical limb ischemia, potentially resulting in better outcomes for diabetic patients and preventing lower limb amputations. The encapsulation process also has value in other types of cell therapy, as it could protect cells from host defenses and increase dwell time.

Coadministration of endothelial and smooth muscle cells derived from human induced pluripotent stem cells as a therapy for critical limb ischemia.

Critical limb ischemia is a condition in which tissue necrosis occurs due to arterial occlusion, resulting in limb amputation in severe cases. Both endothelial cells (ECs) and vascular smooth muscle cells (SMCs) are needed for the regeneration of peripheral arteries in ischemic tissues. However, it is difficult to isolate and cultivate primary EC and SMC from patients for therapeutic angiogenesis. Induced pluripotent stem cells (iPSCs) are regarded as useful stem cells due to their pluripotent differentiation potential. In this study, we explored the therapeutic efficacy of human iPSC‐derived EC and iPSC‐derived SMC in peripheral artery disease model. After the induction of mesodermal differentiation of iPSC, CD34+ progenitor cells were isolated by magnetic‐activated cell sorting. Cultivation of the CD34+ progenitor cells in endothelial culture medium induced the expression of endothelial markers and phenotypes. Moreover, the CD34+ cells could be differentiated into SMC by cultivation in SMC culture medium. In a murine hindlimb ischemia model, cotransplantation of EC with SMC improved blood perfusion and increased the limb salvage rate in ischemic limbs compared to transplantation of either EC or SMC alone. Moreover, cotransplantation of EC and SMC stimulated angiogenesis and led to the formation of capillaries and arteries/arterioles in vivo. Conditioned medium derived from SMC stimulated the migration, proliferation, and tubulation of EC in vitro, and these effects were recapitulated by exosomes isolated from the SMC‐conditioned medium. Together, these results suggest that iPSC‐derived SMC enhance the therapeutic efficacy of iPSC‐derived EC in peripheral artery disease via an exosome‐mediated paracrine mechanism.

Contemporary Endovascular Outcomes for Critical Limb Ischemia Are Still Failing to Meet Society for Vascular Surgery Objective Performance Goals.

The Society for Vascular Surgery (SVS) created Objective Performance Goals (OPGs) for critical limb ischemia (CLI) in 2009. It was previously shown that endovascular therapy for CLI was not meeting these benchmarks. The OPG for all peripheral interventions is <8% for major adverse cardiac events (MACE), <8% for major adverse limb events (MALE), and <3% for major amputation. The goal of this study is to evaluate if outcomes have improved for CLI in recent years, specifically 2015-2018. The Targeted Vascular Module from the American College of Surgeons National Surgical Quality Improvement Program (NSQIP) was queried to identify patients who underwent endovascular intervention for critical limb ischemia from 2011-2018. Cohorts were divided into 2011-2014 and 2015-2018. Primary 30-day outcomes were MACE, MALE, and major amputation. Univariate analyses were performed using the Fisher's exact test and the Wilcoxon rank-sum test. Multivariate analysis comparing groups was performed using inverse probability weights and trend over time analysis was performed using logistic regression with year of intervention as a continuous variable. From 2011 to 2018, 7,168 patients underwent an endovascular intervention for CLI. 28% were classified as "OPG high anatomic risk," and 17% were classified as "OPG high clinical risk." The 2015-2018 cohort vs. the 2011-14 cohort experienced MACE in 3.3% vs. 2.7% (p = .23), MALE in 9.1% vs. 8.9% (p = 0.83), and amputation in 4.0% vs. 4.2% (p = 0.71). When only high anatomic risk patients were considered (n = 1988), MACE was experienced in 2.4% vs. 2.2% (p = 0.87), MALE by 9.5% vs. 10.6% (p = 0.47) and amputation by 5.1% vs. 6.0% (p = 0.40). When only high clinical risk patients were considered (n = 1224), MACE was experienced in 5.2% vs. 3.9% (p = 0.33), MALE by 8.0% vs. 7.4% (p = 0.74) and amputation by 3.9% vs. 3.7% (p = 0.88). Comparing 2015-2018 to the reference 2011-2014, MALE adjusted odds ratio (AOR) = 0.99, 95% CI [0.83-1.18], MACE AOR = 1.19 95% CI [0.88-1.60], and major amputation AOR = 0.91 95% CI [0.70-1.17]. There were no decreases in the trend over time for MALE (AOR per year 0.97, CI [.94-1.02], major amputation (AOR per year: 0.97, CI [0.91-1.03], nor for MACE (AOR per year: 1.05, CI [.98-1.13]). Outcomes following endovascular interventions for CLI continue to underperform when compared to OPG benchmarks for MALE and amputations. There is no decrease over time for these target outcomes. Target MACE events remain acceptable despite the increasing clinical complexity of patients being treated.

Double VEGF/HGF Gene Therapy in Critical Limb Ischemia Complicated by Diabetes Mellitus

Critical leg ischemia (CLI) complicated by diabetes mellitus (DM), which is a very common and dangerous disease, represents the ultimate stage of peripheral arterial disease. Patients are treated with antiplatelet drugs, statins and limb revascularization, but a significant number of patients are not candidate for revascularization. Literature shows that in such cases, gene therapy could be a perfect therapeutic option. The aim of our study was to evaluate efficacy of double vascular endothelial growth factor/hepatocyte growth factor (VEGF/HGF) gene therapy in patients with CLI complicated by DM. We observed that 90 days after administration, serum level of VEGF and ankle-brachial index increased significantly (p < 0.001) and rest pain decreased significantly compared with the control group (p < 0.002). Moreover considerable improvement in vascularization was observed in computed tomography angiography (P = 0.04). Based on the results of this study, we suggest that the therapy with pIRES/VEGF165/HGF bicistronic plasmid administration is a safe and effective method of treatment of patients with both CLI and DM.Graphical abstract

Progress of Gene Therapy in Cardiovascular Disease.

Gene therapy has been extensively studied in peripheral and cardiac ischemia, heart and vein graft failure, and dyslipidemia, but most clinical trials failed to show their efficacies despite good outcomes in preclinical studies. So far, 2 gene therapies for dyslipidemia and one for critical limb ischemia in peripheral artery disease have been approved. In critical limb ischemia, gene therapy using proangiogenic factors has emerged as a novel therapeutic modality for promoting angiogenesis. Initial researches mainly focused on vascular endothelial growth factor, fibroblast growth factor, or hepatocyte growth factor. After the favorable results of basic research, several phase I and II clinical trials of these proangiogenic factors have shown promising results. However, only a phase III clinical trial of the intramuscular injection of hepatocyte growth factor plasmid DNA has shown successful outcomes, and it was recently approved in Japan for treating patients with critical limb ischemia who have ulcers and for whom no alternative therapeutic options are available. DNA vaccine is another promising modality of gene therapy. An antitumor vaccine suppressing angiogenesis through the inhibition of proangiogenic factors and an antihypertensive vaccine inhibiting the renin-angiotensin system are representative DNA vaccines. The advantage of DNA vaccine is its long-term effectiveness with a few vaccinations; however, the benefits and risks, such as adverse T-cell reaction against self-antigen or long-term side effects, of DNA vaccines should be carefully evaluated. In this review, we discuss the recent advances in proangiogenic gene therapy for critical limb ischemia and DNA vaccine for hypertension.