Model Of Critical Limb Ischemia Research Articles

Cell-mediated delivery of VEGF modified mRNA enhances blood vessel regeneration and ameliorates murine critical limb ischemia

Synthetic chemically modified mRNAs (modRNA) encoding vascular endothelial growth factor (VEGF) represents an alternative to gene therapy for the treatment of ischemic cardiovascular injuries. However, novel delivery approaches of modRNA are needed to improve therapeutic efficacy in the diseased setting. We hypothesized that cell-mediated modRNA delivery may enhance the in vivo expression kinetics of VEGF protein thus promoting more potent angiogenic effects. Here, we employed skin fibroblasts as a “proof of concept” to probe the therapeutic potential of a cell-mediated mRNA delivery system in a murine model of critical limb ischemia (CLI). We show that fibroblasts pre-treated with VEGF modRNA have the potential to fully salvage ischemic limbs. Using detailed molecular analysis we reveal that a fibroblast-VEGF modRNA combinatorial treatment significantly reduced tissue necrosis and dramatically improved vascular densities in CLI-injured limbs when compared to control and vehicle groups. Furthermore, fibroblast-delivered VEGF modRNA treatment increased the presence of Pax7+ satellite cells, indicating a possible correlation between VEGF and satellite cell activity. Our study is the first to demonstrate that a cell-mediated modRNA therapy could be an alternative advanced strategy for cardiovascular diseases.

Assessing Therapeutic Angiogenesis in a Murine Model of Hindlimb Ischemia.

Critical limb ischemia (CLI) is a serious condition that entails a high risk of lower limb amputation. Despite revascularization being the gold-standard therapy, a considerable number of CLI patients are not suited for either surgical or endovascular revascularization. Angiogenic therapies are emerging as an option for these patients but are currently still under investigation. Before application in humans, those therapies must be tested in animal models and its mechanisms must be clearly understood. An animal model of hindlimb ischemia (HLI) has been developed by the ligation and excision of the distal external iliac and femoral arteries and veins in mice. A comprehensive panel of tests was assembled to assess the effects of ischemia and putative angiogenic therapies at functional, histologic and molecular levels. Laser Doppler was used for the flow measurement and functional assessment of perfusion. Tissue response was evaluated by the analysis of capillary density after staining with the anti-CD31 antibody on histological sections of gastrocnemius muscle and by measurement of collateral vessel density after diaphonization. Expression of angiogenic genes was quantified by RT-PCR targeting selected angiogenic factors exclusively in endothelial cells (ECs) after laser capture microdissection from mice gastrocnemius muscles. These methods were sensitive in identifying differences between ischemic and non-ischemic limbs and between treated and non-treated limbs. This protocol provides a reproducible model of CLI and a framework for testing angiogenic therapies.

Abstract 17039: Neutrophil Subset in Blood Contributes to Acute Oxidant Signals in the Bone Marrow, Which Regulate Innate Immune Responses After Ischemic Tissue Damage

Blood neutrophils are short-lived and have distinct subsets. Aged (senescent) neutrophils are cleared by macrophages in multiple organs including bone marrow (BM), at least, in the steady state. It is unknown, after ischemic tissue damage, how these aged-type neutrophils regulate bone marrow microenvironment and impact on downstream bone marrow responses. In a mouse model of critical limb ischemia, we found that CD62L lo aged-type neutrophils that return to the BM, measured by in vivo circulating cell-labeling, are increased (2.1-fold) after acute limb ischemia, which is corresponded with acute increase in CXCR4, a major chemokine receptor promoting BM returning of blood neutrophils (31%). Interestingly, neutrophil uptake by macrophages is reduced (by 76%) in the BM after limb ischemia, suggesting increased retention of blood-derived CD62L lo neutrophils in BM microenvironment after ischemia. We also found, in the BM, that blood-derived neutrophils have higher reactive oxygen species (ROS) than other neutrophils (61% in MFI) and that limb ischemia indeed increases ROS (34%) and oxidized lipid microparticles (61%) in BM plasma in a Nox2 NADPH oxidase-dependent manner. These suggest that increased returning and retention of blood-derived CD62L lo neutrophils in the BM may contribute to oxidant signals in the BM after limb ischemia. Indeed, using aged neutrophils generated ex vivo and adoptive transfer of these cells, we found that aged neutrophils modulate BM innate immune responses after limb ischemia through ROS—reduced activation of hematopoietic stem progenitor cells (50%); increased acute activation of myeloid progenitor cells (4%); and increased acute but inhibited later Ly6C hi inflammatory monocytes/macrophages in the ischemic tissue (72% and 45%, respectively)—resulting in improved tissue recovery from ischemic damage (2.3-fold). In summary, an aged-type neutrophil subset in blood contributes to acute oxidant signals in the BM, which coordinate innate immune responses to promote tissue recovery from ischemic damage.

Restoring Endothelial Function by Targeting Desert Hedgehog Downstream of Klf2 Improves Critical Limb Ischemia in Adults.

Klf (kruppel-like factor) 2 is critical to establish and maintain endothelial integrity. Therefore, determining upstream and downstream mediators of Klf2 would lead to alternative therapeutic targets in cardiovascular disease management. Here we identify Dhh (desert hedgehog) as a downstream effector of Klf2, whose expression in endothelial cells (ECs) is upregulated by shear stress and decreased by inflammatory cytokines. Consequently, we show that Dhh knockdown in ECs promotes endothelial permeability and EC activation and that Dhh agonist prevents TNF-α (tumor necrosis factor alpha) or glucose-induced EC dysfunction. Moreover, we demonstrate that human critical limb ischemia, a pathological condition linked to diabetes mellitus and inflammation, is associated to major EC dysfunction. By recreating a complex model of critical limb ischemia in diabetic mice, we found that Dhh-signaling agonist significantly improved EC function without promoting angiogenesis, which subsequently improved muscle perfusion. Restoring EC function leads to significant critical limb ischemia recovery. Dhh appears to be a promising target, downstream of Klf2, to prevent the endothelial dysfunction involved in ischemic vascular diseases.

Serum C-peptide level correlates with the course of muscle tissue healing in the rabbit model of critical limb ischemia.

The therapeutic potential of adipose-derived stem cell conditioned medium (ASC-CM) was studied in the rabbit model of critical limb ischemia (CLI). Rabbits received treatment with ASC-CM or placebo. Gastrocnemius muscle tissue was collected 35 days after ischemia induction. Ischemic changes were evaluated in hematoxylin-eosin stained tissues for early (necrotic lesions/granulation tissue) and late (fibrous scars) phases of tissue repair. The expression of proangiogenic miR-126 was also evaluated using in situ hybridization. The levels of cytokines, insulin, and C-peptide were measured in blood. Early repair phases were observed more often in placebo-treated samples (45.5%) than in ASC-CM-treated ones (22.2%). However, the difference was not statistically significant. We demonstrated a statistically significant positive correlation between the early healing phases in tissue samples and C-peptide levels in peripheral blood. The expression of proangiogenic miR-126 was also shown in a number of structures in all phases of ischemic tissue healing. Based on our results, we believe that treatment with ASC-CM has the potential to accelerate the healing process in ischemic tissues in the rabbit model of CLI. The whole healing process was accompanied by miR-126 tissue expression. C-peptide could be used to monitor the course of the tissue healing process.

Invited commentary

A major limitation in developing molecular therapies for critical limb ischemia has been the lack of a clinically relevant model to test efficacy. The existing animal models primarily depend on acute limb ischemia generated by femoral artery ligation or excision. These models have provided insights into cellular and molecular mechanisms that underlie collateral artery growth, primarily because young rodents collateralize quickly in response to ischemia. This trait is less helpful when trying to test potential treatments that will be effective in older, atherosclerotic, diabetic human patients. Many single growth factor therapies have showed efficacy in these rodent-based models, only to fail in human clinical trials. Furthermore, these surgical models require invasive isolation of the femoral artery, creating an inflammatory milieu for arteriogenesis different from that caused by atherosclerosis. They also lack the tibial level occlusions seen in diabetic patients. The current article addresses the last two concerns. The authors have modified the model developed by Liddell et al,1Liddell R.P. Patel T.H. Weiss C.R. Lee D.S. Matsuhashi T. Browh P.R. et al.Endovascular model of rabbit hindlimb ischemia: a platforms to evaluate therapeutic angiogenesis.J Vasc Interv Radiol JVIR. 2005; 16: 991-998Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar where the authors used a percutaneous transauricular approach to coil embolize the rabbit superficial femoral artery, by instead using 300- to 500-μm microspheres to embolize more distal targets: the saphenous (posterior tibial equivalent) and anterior tibial arteries and their outflow beds. The resulting acute ischemia resulted in significant hindlimb ulcerations, similar to the pedal gangrene seen in patients with critical limb ischemia. The ulcerations and decreased saphenous artery blood flow were persistent up to 14 days at the time of humane killing. It is likely that this model will need further refinement to be made practical for use in testing arteriogenic therapies. The embolization resulted in a significant acute ischemic injury to the muscle, with a large inflammatory cell infiltrate and muscle necrosis. The footpad necrosis in most of the rabbits was severe, with high pain scores, resulting in ethical concerns for maintaining the model for longer than 14 days. Staged embolizations of the anterior tibial and saphenous arteries with particles sized for the tibial arteries rather than their outflow beds might better reflect the tibial level occlusions seen in diabetic humans and provide a less severe and more chronic ischemia that would better support therapeutic testing. This method would also facilitate use of the model in hypercholesterolemic or diabetic rabbits, who would presumably be less tolerant of the severe arterial ischemic insult generated by the current protocol. Limitations aside, this percutaneous embolization-based model of hindlimb ischemia represents a step forward in the creation of a more relevant animal model of critical limb ischemia. It remains to be seen whether it is enough of a step forward to support robust preclinical testing of arteriogenic therapies. Evaluation of a new model of hind limb ischemia in rabbitsJournal of Vascular SurgeryVol. 68Issue 3PreviewVarious animal models of critical limb ischemia have been developed in the past. However, there is no animal model that can undergo endovascular treatment, while providing reproducible true critical limb ischemia with arterial ulcers and rest pain. We evaluated the efficacy of a new model of rabbit hindlimb ischemia created through a percutaneous approach using embolization with calibrated particles. Full-Text PDF Open Archive

The controversial origin of pericytes during angiogenesis - Implications for cell-based therapeutic angiogenesis and cell-based therapies.

Pericytes reside within the basement membrane of small vessels and are often in direct cellular contact with endothelial cells, fulfilling important functions during blood vessel formation and homeostasis. Recently, these pericytes have been also identified as mesenchymal stem cells. Mesenchymal stem cells, and especially their specialized subpopulation of pericytes, represent promising candidates for therapeutic angiogenesis applications, and have already been widely applied in pre-clinical and clinical trials. However, cell-based therapies of ischemic diseases (especially of myocardial infarction) have not resulted in significant long-term improvement. Interestingly, pericytes from a hematopoietic origin were observed in embryonic skin and a pericyte sub-population expressing leukocyte and monocyte markers was described during adult angiogenesis in vivo. Since mesenchymal stem cells do not express hematopoietic markers, the latter cell type might represent an alternative pericyte population relevant to angiogenesis. Therefore, we sourced blood-derived angiogenic cells (BDACs) from monocytes that closely resembled hematopoietic pericytes, which had only been observed in vivo thus far. BDACs displayed many pericytic features and exhibited enhanced revascularization and functional tissue regeneration in a pre-clinical model of critical limb ischemia. Comparison between BDACs and mesenchymal pericytes indicated that BDACs (while resembling hematopoietic pericytes) enhanced early stages of angiogenesis, such as endothelial cell sprouting. In contrast, mesenchymal pericytes were responsible for blood vessel maturation and homeostasis, while reducing endothelial sprouting.Since the formation of new blood vessels is crucial during therapeutic angiogenesis or during integration of implants into the host tissue, hematopoietic pericytes (and therefore BDACs) might offer an advantageous addition or even an alternative for cell-based therapies.

Hypoxia and Ischemia Promote a Maladaptive Platelet Phenotype.

Reduced blood flow and tissue oxygen tension conditions result from thrombotic and vascular diseases such as myocardial infarction, stroke, and peripheral vascular disease. It is largely assumed that while platelet activation is increased by an acute vascular event, chronic vascular inflammation, and ischemia, the platelet activation pathways and responses are not themselves changed by the disease process. We, therefore, sought to determine whether the platelet phenotype is altered by hypoxic and ischemic conditions. In a cohort of patients with metabolic and peripheral artery disease, platelet activity was enhanced, and inhibition with oral antiplatelet agents was impaired compared with platelets from control subjects, suggesting a difference in platelet phenotype caused by the disease. Isolated murine and human platelets exposed to reduced oxygen (hypoxia chamber, 5% O2) had increased expression of some proteins that augment platelet activation compared with platelets in normoxic conditions (21% O2). Using a murine model of critical limb ischemia, platelet activity was increased even 2 weeks postsurgery compared with sham surgery mice. This effect was partly inhibited in platelet-specific ERK5 (extracellular regulated protein kinase 5) knockout mice. These findings suggest that ischemic disease changes the platelet phenotype and alters platelet agonist responses because of changes in the expression of signal transduction pathway proteins. Platelet phenotype and function should, therefore, be better characterized in ischemic and hypoxic diseases to understand the benefits and limitations of antiplatelet therapy.

A Novel Hydrogen Sulfide Prodrug, SG-1002, Augments Angiogenesis and Coronary Vascular Tone in a Swine Model of Critical Limb Ischemia

A Novel Hydrogen Sulfide Prodrug, SG-1002, Augments Angiogenesis and Coronary Vascular Tone in a Swine Model of Critical Limb Ischemia

Deletion of TXNIP Mitigates High-Fat Diet-Impaired Angiogenesis and Prevents Inflammation in a Mouse Model of Critical Limb Ischemia.

Background: Previous work demonstrated that high-fat diet (HFD) triggered thioredoxin-interacting protein (TXNIP) and that silencing TXNIP prevents diabetes-impaired vascular recovery. Here, we examine the impact of genetic deletion of TXNIP on HFD-impaired vascular recovery using hind limb ischemia model. Methods: Wild type mice (WT, C57Bl/6) and TXNIP knockout mice (TKO) were fed either normal chow diet (WT-ND and TKO-ND) or 60% high-fat diet (WT-HFD and TKO-HFD). After four weeks of HFD, unilateral hind limb ischemia was performed and blood flow was measured using Laser doppler scanner at baseline and then weekly for an additional three weeks. Vascular density, nitrative stress, infiltration of CD68+ macrophages, and expression of inflammasome, vascular endothelial growth factor (VEGF), VEGF receptor-2 were examined by slot blot, Western blot and immunohistochemistry. Results: By week 8, HFD caused similar increases in weight, cholesterol and triglycerides in both WT and TKO. At week 4 and week 8, HFD significantly impaired glucose tolerance in WT and to a lesser extent in TKO. HFD significantly impaired blood flow and vascular density (CD31 labeled) in skeletal muscle of WT mice compared to ND but not in TKO. HFD and ischemia significantly induced tyrosine nitration, and systemic IL-1β and infiltration of CD68+ cells in skeletal muscle from WT but not from TKO. HFD significantly increased cleaved-caspase-1 and IL-1 β compared to ND. Under both ND, ischemia tended to increase VEGF expression and increased VEGFR2 activation in WT only but not TKO. Conclusion: Similar to prior observation in diabetes, HFD-induced obesity can compromise vascular recovery in response to ischemic insult. The mechanism involves increased TXNIP-NLRP3 (nucleotide-binding oligomerization domain-like receptor protein 3) inflammasome activation, nitrative stress and impaired VEGFR2 activation. Deletion of TXNIP restored blood flow, reduced nitrative stress and blunted inflammasome-mediated inflammation; however, it did not impact VEGF/VEGFR2 in HFD. Targeting TXNIP-NLRP3 inflammasome can provide potential therapeutic target in obesity-induced vascular complication.

Abstract 150: Hypoxia and Ischemia Promote a Maladaptive Platelet Phenotype

Background: An understudied area of platelet biology is that ischemic diseases such as peripheral vascular disease (PVD) can alter the platelet phenotype, and this may lead to unpredictable effects of anti-platelet agents. Methods: Isolated murine and human platelets were exposed to a reduced oxygen environment (hypoxia chamber, 5% O 2 ). Platelet activation was assessed as mean fluorescence intensity (MFI) of surface p-selectin by flow cytometry, and morphologic changes by confocal microscopy. A murine model of critical limb ischemia (CLI) was used to assess changes in platelet activity in wild-type (WT) and in platelet ERK5 -/- mice. Limb blood flow was assessed by laser Doppler imaging. Hypoxia was additionally examined in mice by unilateral pneumonectomy. Western blotting was used to assess protein expression. Results: In human platelets ex vivo at 5% O 2 , platelet activation increased to 5076 MFI±409 from 3548 MFI±187 at 20% O 2 (P=0.005). With the ERK5 inhibitor XMD892, platelet activation at 5% O 2 decreased from 5677 MFI±312 to 3175 MFI±247 (p=0.001). In a murine model of PVD, enhanced platelet activation was sustained for weeks vs. sham, and limb blood flow in platelet ERK5 -/- mice one week after ischemia was augmented by 26%: 0.61±0.03 vs 0.87±0.03 vs, WT mice (p=0.0002), with 50% less platelet activity. In mice with unilateral pneumonectomy vs. sham surgery, observed hypoxia was accompanied by increased platelet ERK5 activation coincident with enhanced platelet activation. In patients with CLI, augmented platelet activation in spite of aspirin treatment was observed via the thromboxane and the PAR1 receptors, and especially via P2Y 12 receptor. Conclusions: Hypoxia and ischemic tissue injury, such as with critical limb ischemia, change the phenotype of the platelet, promoting a pro-thrombotic state that is partly dependent on platelet ERK5. Platelet phenotype and function should be better characterized in ischemic diseases.

PET Cell Tracking Using 18F-FLT is Not Limited by Local Reuptake of Free Radiotracer

Assessing the retention of cell therapies following implantation is vital and often achieved by labelling cells with 2′-[18F]-fluoro-2′-deoxy-D-glucose (18F-FDG). However, this approach is limited by local retention of cell-effluxed radiotracer. Here, in a preclinical model of critical limb ischemia, we assessed a novel method of cell tracking using 3′-deoxy-3′-L-[18F]-fluorothymidine (18F-FLT); a clinically available radiotracer which we hypothesise will result in minimal local radiotracer reuptake and allow a more accurate estimation of cell retention. Human endothelial cells (HUVECs) were incubated with 18F-FDG or 18F-FLT and cell characteristics were evaluated. Dynamic positron emission tomography (PET) images were acquired post-injection of free 18F-FDG/18F-FLT or 18F-FDG/18F-FLT-labelled HUVECs, following the surgical induction of mouse hind-limb ischemia. In vitro, radiotracer incorporation and efflux was similar with no effect on cell viability, function or proliferation under optimised conditions (5 MBq/mL, 60 min). Injection of free radiotracer demonstrated a faster clearance of 18F-FLT from the injection site vs. 18F-FDG (p ≤ 0.001), indicating local cellular uptake. Using 18F-FLT-labelling, estimation of HUVEC retention within the engraftment site 4 hr post-administration was 24.5 ± 3.2%. PET cell tracking using 18F-FLT labelling is an improved approach vs. 18F-FDG as it is not susceptible to local host cell reuptake, resulting in a more accurate estimation of cell retention.

Marrow-isolated adult multilineage inducible cells embedded within a biologically-inspired construct promote recovery in a mouse model of peripheral vascular disease

Peripheral vascular disease is one of the major vascular complications in individuals suffering from diabetes and in the elderly that is associated with significant burden in terms of morbidity and mortality. Stem cell therapy is being tested as an attractive alternative to traditional surgery to prevent and treat this disorder. The goal of this study was to enhance the protective and reparative potential of marrow-isolated adult multilineage inducible (MIAMI) cells by incorporating them within a bio-inspired construct (BIC) made of two layers of gelatin B electrospun nanofibers. We hypothesized that the BIC would enhance MIAMI cell survival and engraftment, ultimately leading to a better functional recovery of the injured limb in our mouse model of critical limb ischemia compared to MIAMI cells used alone. Our study demonstrated that MIAMI cell-seeded BIC resulted in a wide range of positive outcomes with an almost full recovery of blood flow in the injured limb, thereby limiting the extent of ischemia and necrosis. Functional recovery was also the greatest when MIAMI cells were combined with BICs, compared to MIAMI cells alone or BICs in the absence of cells. Histology was performed 28 days after grafting the animals to explore the mechanisms at the source of these positive outcomes. We observed that our critical limb ischemia model induces an extensive loss of muscular fibers that are replaced by intermuscular adipose tissue (IMAT), together with a highly disorganized vascular structure. The use of MIAMI cells-seeded BIC prevented IMAT infiltration with some clear evidence of muscular fibers regeneration.

Improving the therapeutic efficacy of mesenchymal stromal cells to restore perfusion in critical limb ischemia through pulsed focused ultrasound

Mesenchymal stem cells (MSC) are promising therapeutics for critical limb ischemia (CLI). Mechanotransduction from pulsed focused ultrasound (pFUS) upregulates local chemoattractants to enhance homing of intravenously (IV)-infused MSC and improve outcomes. This study investigated whether pFUS exposures to skeletal muscle would improve local homing of iv-infused MSCs and their therapeutic efficacy compared to iv-infused MSCs alone. CLI was induced by external iliac arterial cauterization in 10–12-month-old mice. pFUS/MSC treatments were delayed 14 days, when surgical inflammation subsided. Mice were treated with iv-saline, pFUS alone, IV-MSC, or pFUS and IV-MSC. Proteomic analyses revealed pFUS upregulated local chemoattractants and increased MSC tropism to CLI muscle. By 7 weeks post-treatment, pFUS + MSC significantly increased perfusion and CD31 expression, while reducing fibrosis compared to saline. pFUS or MSC alone reduced fibrosis, but did not increase perfusion or CD31. Furthermore, MSCs homing to pFUS-treated CLI muscle expressed more vascular endothelial growth factor (VEGF) and interleukin-10 (IL-10) than MSCs homing to non-pFUS-treated muscle. pFUS + MSC improved perfusion and vascular density in this clinically-relevant CLI model. The molecular effects of pFUS increased both MSC homing and MSC production of VEGF and IL-10, suggesting microenvironmental changes from pFUS also increased potency of MSCs in situ to further enhance their efficacy.

414 - Deletion of TXNIP Mitigates High Fat Diet-Impaired Blood Flow and Inflammation in a Model of Critical Limb Ischemia

Introduction Previous work demonstrated that silencing thioredoxin-interacting protein (TXNIP) prevents diabetes-impaired vascular recovery in diabetic model. Here, we examine the impact of high fat diet (HFD) on impaired vascular recovery and possible protective roles of deleting TXNIP. We hypothesize that deletion of TXNIP prevents HFD-mediated nitrative stress, inflammation and preserves vascular recovery. Methods Wild type mice (WT, C57Bl/6) and TXNIP knockout mice (TKO) were fed either normal chow (WT-ND and TKO-ND) or 60% fat diet (WT-HFD and TKO-HFD). After 4 weeks of HFD, unilateral hind limb ischemia was performed and blood flow was measured using Laser doppler scanner at baseline and then weekly for additional 3 weeks. Vascular density, nitrative stress, infiltration of CD68-macrophages, and expression of inflammasome, VEGF, VEGF receptor-2 were examined by slot blot, Western blot and immunohistochemistry. Results HFD increased total body weight and induced insulin resistance in both WT and TKO mice. HFD significantly impaired blood flow and vascular density (CD31 labeled) in skeletal muscle of WT mice compared to ND but not in TKO. HFD and ischemia induced tyrosine nitration, cleaved caspase-1 and IL-1 and infiltration of CD68 in skeletal muscle from WT but not from TKO. HFD or ischemia had no effect on VEGF or VEGFR2 levels in all groups. Under ND, ischemia induced phosphorylation of VEGFR2 in both WT and TKO. HFD impaired VEGFR2 activation in WT but not in TKO. Conclusion Similar to diabetes, HFD-induced obesity can compromise vascular recovery in response to ischemic insult. The mechanism involves increased nitrative stress and impaired VEGFR2 activation. Deletion of TXNIP reduced nitrative stress and restored VEGFR2 signaling and blood flow in HFD. Targeting TXNIP can provide potential therapeutic target in obesity-induced vascular complication.

Therapeutic Potential of Adipose-Derived Therapeutic Factor Concentrate for Treating Critical Limb Ischemia.

Transplantation of adipose-derived stem cells (ADSCs) is an emerging therapeutic option for addressing intractable diseases such as critical limb ischemia (CLI). Evidence suggests that therapeutic effects of ADSCs are primarily mediated through paracrine mechanisms rather than transdifferentiation. These secreted factors can be captured in conditioned medium (CM) and concentrated to prepare a therapeutic factor concentrate (TFC) composed of a cocktail of beneficial growth factors and cytokines that individually and in combination demonstrate disease-modifying effects. The ability of a TFC to promote reperfusion in a rabbit model of CLI was evaluated. A total of 27 adult female rabbits underwent surgery to induce ischemia in the left hindlimb. An additional five rabbits served as sham controls. One week after surgery, the ischemic limbs received intramuscular injections of either (1) placebo (control medium), (2) a low dose of TFC, or (3) a high dose of TFC. Limb perfusion was serially assessed with a Doppler probe. Blood samples were analyzed for growth factors and cytokines. Tissue was harvested postmortem on day 35 and assessed for capillary density by immunohistochemistry. At 1 month after treatment, tissue perfusion in ischemic limbs treated with a high dose of TFC was almost double (p < 0.05) that of the placebo group [58.8 ± 23 relative perfusion units (RPU) vs. 30.7 ± 13.6 RPU; mean ± SD]. This effect was correlated with greater capillary density in the affected tissues and with transiently higher serum levels of the angiogenic and prosurvival factors vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF). The conclusions from this study are that a single bolus administration of TFC demonstrated robust effects for promoting tissue reperfusion in a rabbit model of CLI and that a possible mechanism of revascularization was promotion of angiogenesis by TFC. Results of this study demonstrate that TFC represents a potent therapeutic cocktail for patients with CLI, many of whom are at risk for amputation of the affected limb.

Glyoxalase-1 Overexpression Reverses Defective Proangiogenic Function of Diabetic Adipose-Derived Stem Cells in Streptozotocin-Induced Diabetic Mice Model of Critical Limb Ischemia.

Adipose‐derived stem cell (ADSC)‐based therapy is promising for critical limb ischemia (CLI) treatment, especially in patients with diabetes. However, the therapeutic effects of diabetic ADSCs (D‐ADSCs) are impaired by the diabetes, possibly through intracellular reactive oxygen species (ROS) accumulation. The objective of the present study was to detect whether overexpression of methylglyoxal‐metabolizing enzyme glyoxalase‐1 (GLO1), which reduces ROS in D‐ADSCs, can restore their proangiogenic function in a streptozotocin‐induced diabetic mice model of CLI. GLO1 overexpression in D‐ADSCs (G‐D‐ADSCs) was achieved using the lentivirus method. G‐D‐ADSCs showed a significant decrease in intracellular ROS accumulation, increase in cell viability, and resistance to apoptosis under high‐glucose conditions compared with D‐ADSCs. G‐D‐ADSCs also performed better in terms of migration, differentiation, and proangiogenic capacity than D‐ADSCs in a high‐glucose environment. Notably, these properties were restored to the same level as that of nondiabetic ADSCs under high‐glucose conditions. G‐D‐ADSC transplantation induced improved reperfusion and an increased limb salvage rate compared D‐ADSCs in a diabetic mice model of CLI. Histological analysis revealed higher microvessel densities and more G‐D‐ADSC‐incorporated microvessels in the G‐D‐ADSC group than in the D‐ADSC group, which was comparable to the nondiabetic ADSC group. Higher expression of vascular endothelial growth factor A and stromal cell‐derived factor‐1α and lower expression of hypoxia‐induced factor‐1α were also detected in the ischemic muscles from the G‐D‐ADSC group than that of the D‐ADSC group. The results of the present study have demonstrated that protection from ROS accumulation by GLO1 overexpression is effective in reversing the impaired biological function of D‐ADSCs in promoting neovascularization of diabetic CLI mice model and warrants the future clinical application of D‐ADSC‐based therapy in diabetic patients. Stem Cells Translational Medicine 2017;6:261–271

Profound Actions of an Agonist of Growth Hormone-Releasing Hormone on Angiogenic Therapy by Mesenchymal Stem Cells.

The efficiency of cell therapy is limited by poor cell survival and engraftment. Here, we studied the effect of the growth hormone-releasing hormone agonist, JI-34, on mesenchymal stem cell (MSC) survival and angiogenic therapy in a mouse model of critical limb ischemia. Mouse bone marrow-derived MSCs were incubated with or without 10(-8) mol/L JI-34 for 24 hours. MSCs were then exposed to hypoxia and serum deprivation to detect the effect of preconditioning on cell apoptosis, migration, and tube formation. For in vivo tests, critical limb ischemia was induced by femoral artery ligation. After surgery, mice received 50 μL phosphate-buffered saline or with 1×10(6) MSCs or with 1×10(6) JI-34-reconditioned MSCs. Treatment of MSCs with JI-34 improved MSC viability and mobility and markedly enhanced their capability to promote endothelial tube formation in vitro. These effects were paralleled by an increased phosphorylation and nuclear translocation of signal transducer and activator of transcription 3. In vivo, JI-34 pretreatment enhanced the engraftment of MSCs into ischemic hindlimb muscles and augmented reperfusion and limb salvage compared with untreated MSCs. Significantly more vasculature and proliferating CD31(+) and CD34(+) cells were detected in ischemic muscles that received MSCs treated with JI-34. Our studies demonstrate a novel role for JI-34 to markedly improve therapeutic angiogenesis in hindlimb ischemia by increasing the viability and mobility of MSCs. These findings support additional studies to explore the full potential of growth hormone-releasing hormone agonists to augment cell therapy in the management of ischemia.

Muscle Derived Stem Cells Stimulate Muscle Myofiber Repair and Counteract Fat Infiltration in a Diabetic Mouse Model of Critical Limb Ischemia.

BackgroundCritical Limb Ischemia (CLI) affects patients with Type 2 Diabetes (T2D) and obesity, with high risk of amputation and post-surgical mortality, and no effective medical treatment. Stem cell therapy, mainly with bone marrow mesenchymal, adipose derived, endothelial, hematopoietic, and umbilical cord stem cells, is promising in CLI mouse and rat models and is in clinical trials. Their general focus is on angiogenic repair, with no reports on the alleviation of necrosis, lipofibrosis, and myofiber regeneration in the ischemic muscle, or the use of Muscle Derived Stem Cells (MDSC) alone or in combination with pharmacological adjuvants, in the context of CLI in T2D.MethodsUsing a T2D mouse model of CLI induced by severe unilateral femoral artery ligation, we tested: a) the repair efficacy of MDSC implanted into the ischemic muscle and the effects of concurrent intraperitoneal administration of a nitric oxide generator, molsidomine; and b) whether MDSC may partially counteract their own repair effects by stimulating the expression of myostatin, the main lipofibrotic agent in the muscle and inhibitor of muscle mass.ResultsMDSC: a) reduced mortality, and b) in the ischemic muscle, increased stem cell number and myofiber central nuclei, reduced fat infiltration, myofibroblast number, and myofiber apoptosis, and increased smooth muscle and endothelial cells, as well as neurotrophic factors. The content of myosin heavy chain 2 (MHC-2) myofibers was not restored and collagen was increased, in association with myostatin overexpression. Supplementation of MDSC with molsidomine failed to stimulate the beneficial effects of MDSC, except for some reduction in myostatin overexpression. Molsidomine given alone was rather ineffective, except for inhibiting apoptosis and myostatin overexpression.ConclusionsMDSC improved CLI muscle repair, but molsidomine did not stimulate this process. The combination of MDSC with anti-myostatin approaches should be explored to restore myofiber MHC composition.

Ghrelin Promotes Functional Angiogenesis in a Mouse Model of Critical Limb Ischemia Through Activation of Proangiogenic MicroRNAs.

Current therapeutic strategies for the treatment of critical limb ischemia (CLI) have only limited success. Recent in vitro evidence in the literature, using cell lines, proposes that the peptide hormone ghrelin may have angiogenic properties. In this study, we aim to investigate if ghrelin could promote postischemic angiogenesis in a mouse model of CLI and, further, identify the mechanistic pathway(s) that underpin ghrelin's proangiogenic properties. CLI was induced in male CD1 mice by femoral artery ligation. Animals were then randomized to receive either vehicle or acylated ghrelin (150 μg/kg sc) for 14 consecutive days. Subsequently, synchrotron radiation microangiography was used to assess hindlimb perfusion. Subsequent tissue samples were collected for molecular and histological analysis. Ghrelin treatment markedly improved limb perfusion by promoting the generation of new capillaries and arterioles (internal diameter less than 50 μm) within the ischemic hindlimb that were both structurally and functionally normal; evident by robust endothelium-dependent vasodilatory responses to acetylcholine. Molecular analysis revealed that ghrelin's angiogenic properties were linked to activation of prosurvival Akt/vascular endothelial growth factor/Bcl-2 signaling cascade, thus reducing the apoptotic cell death and subsequent fibrosis. Further, ghrelin treatment activated proangiogenic (miR-126 and miR-132) and antifibrotic (miR-30a) microRNAs (miRs) while inhibiting antiangiogenic (miR-92a and miR-206) miRs. Importantly, in vitro knockdown of key proangiogenic miRs (miR-126 and miR-132) inhibited the angiogenic potential of ghrelin. These results therefore suggest that clinical use of ghrelin for the early treatment of CLI may be a promising and potent inducer of reparative vascularization through modulation of key molecular factors.