Transplantation of Human Embryonic Stem Cell-Derived Pericyte-Like Cells Transduced with Basic Fibroblast Growth Factor Promotes Angiogenic Recovery in Mice with Severe Chronic Hindlimb Ischemia.

Critical limb ischemia (CLI) is a state of severe peripheral artery disease, with no effective treatment. Cell therapy has been investigated as a therapeutic tool for CLI, and pericytes are promising therapeutic candidates based on their angiogenic properties. We firstly generated highly proliferative and immunosuppressive pericyte-like cells from embryonic stem (ES) cells. In order to enhance the angiogenic potential, we transduced the basic fibroblast growth factor (bFGF) gene into the pericyte-like cells and found a significant enhancement of angiogenesis in a Matrigel plug assay. Furthermore, we evaluated the bFGF-expressing pericyte-like cells in the previously established chronic hindlimb ischemia model in which bone marrow-derived MSCs were not effective. As a result, bFGF-expressing pericyte-like cells significantly improved blood flow in both laser Doppler perfusion imaging (LDPI) and dynamic contrast-enhanced MRI (DCE-MRI). These findings suggest that bFGF-expressing pericyte-like cells differentiated from ES cells may be a therapeutic candidate for CLI.

Transplantation of Purified Human Pericytes for Myocardial Repair in SCID-hu Mice.

The physiological, pathological, and regenerative roles of pericytes as microvascular mural cells and multipotent precursors have gained significant attention. The capacity to prospectively purify pericytes from multiple organs enables the investigation of their tissue-specific regenerative capabilities. Here, we describe the application of purified human pericytes for cardiac regeneration post-infarct in an immunodeficient mouse model. This protocol includes experimental details of pericyte isolation from both human skeletal and cardiac muscle, an immunodeficient mouse model of acute myocardial infarction, and xenogeneic pericyte transplantation.

Assessment of Pericyte Phenotype by Flow Cytometry.

Pericytes are mural cells closely associated with endothelial cells in capillaries and microvessels. They are precursors of mesenchymal stem/stromal cells that have historically been retrospectively characterized in culture. We established a protocol, described in this chapter, to characterize and isolate pericytes from multiple human organs by flow cytometry and fluorescence-activated cell sorting. This prospective purification of pericytes brings us a step forward in the development of strategies for their use in the clinic.

Characterization of Human Pericyte Phenotype by Immunohistochemistry.

Pericytes are found in all vascularized organs and are defined anatomically as perivascular cells that closely surround endothelial cells in capillaries and microvessels and are embedded within the same basement membrane. They have been shown to have diverse physiological and pathological functions including regulation of blood pressure, and tissue regeneration and scarring. Fundamental to understanding the role these cells play in these diverse processes is the ability to accurately identify and localize them in vivo. To do this, we have developed multicolor immunohistochemistry protocols described in this chapter.

A Metabolomics-Based Approach to Identify Lineage Guiding Molecules in Pericyte Cultures.

We report the use of self-assembled peptide (F2/S) hydrogels and cellular metabolomics to identify a number of innate molecules that are integral to the metabolic processes which drive cellular differentiation of multipotent pericyte stem cells. The culture system relies solely on substrate mechanics to induce differentiation in the absence of traditional differentiation media and therefore is a non-invasive approach to assessing cellular behavior at the molecular level and identifying key metabolites in this process. This novel approach demonstrates that simple metabolites can provide an alternative means to direct stem cell differentiation and that biomaterials can be used to identify them simply and quickly.

A novel model of chronic limb ischemia to therapeutically evaluate the angiogenic effects of drug candidates.

Critical limb ischemia (CLI) is a severe state of peripheral artery disease with high unmet clinical needs. Further, there are no effective treatment options for patients with CLI. Based on preclinical study results, predicting the clinical efficacy of CLI treatments is typically difficult because conventional hindlimb ischemia (HLI) rodent models display spontaneous recovery from ischemia, which is not observed in patients with CLI. Therefore, we aimed to develop a novel chronic and severe HLI model to properly evaluate the therapeutic effects of drug candidates for CLI. Severe HLI mice (Type-N) were generated by increasing the excised area of blood vessels in a hindlimb of NOG mice. Immunohistochemistry and gene expression analysis at 9 wk after the Type-N operation revealed that the ischemic limb was in a steady state with impaired angiogenesis, like that observed in patients with CLI. We did selection of chronic Type-N mice based on the number of necrotic nails and blood flow rate at 2 wk after surgery because some Type-N mice showed mild symptoms. Therapeutic treatment with cilostazol, which is used for intermittent claudication, did not restore blood flow in chronic Type-N mice. In contrast, therapeutic transplantation of pericytes and vascular endothelial cells, which can form new blood vessels in vivo, significantly improved blood flow in a subset of Type-N mice. These findings suggest that this novel chronic and severe HLI model may be a valuable standard animal model for therapeutic evaluation of the angiogenic effects of CLI drug candidates.NEW & NOTEWORTHY We developed a chronic and severe hindlimb ischemia (HLI) mouse model for preclinical research on critical limb ischemia (CLI). This model partially reflects human CLI pathology in that it does not show spontaneous restoration of blood flow or expression of angiogenic genes in the ischemic limb. This novel model may be valuable for therapeutic evaluation of the angiogenic effects of CLI drug candidates.

Improvement of Neurological Function in Rats with Ischemic Stroke by Adipose-derived Pericytes.

Pericytes possess high multipotent features and cell plasticity, and produce angiogenic and neurotrophic factors that indicate their high regenerative potential. The aim of this study was to investigate whether transplantation of adipose-derived pericytes can improve functional recovery and neurovascular plasticity after ischemic stroke in rats. Rat adipose-derived pericytes were isolated from subcutaneous adipose tissue by fluorescence-activated cell sorting. Adult male Wistar rats were subjected to 90 min of middle cerebral artery occlusion followed by intravenous injection of rat adipose-derived pericytes 24 h later. Functional recovery evaluations were performed at 1, 7, 14, and 28 days after injection of rat adipose-derived pericytes. Angiogenesis and neurogenesis were examined in rat brains using immunohistochemistry. It was observed that intravenous injection of adipose-derived pericytes significantly improved recovery of neurological function in rats with stroke compared to phosphate-buffered saline-treated controls. Immunohistochemical analysis revealed that the number of blood capillaries was significantly increased along the ischemic boundary zone of the cortex and striatum in stroke rats treated with adipose-derived pericytes. In addition, treatment with adipose-derived pericytes increased the number of doublecortin positive neuroblasts. Our data suggest that transplantation of adipose-derived pericytes can significantly improve the neurologic status and contribute to neurovascular remodeling in rats after ischemic stroke. These data provide a new insight for future cell therapies that aim to treat ischemic stroke patients.

Pericytes improve locomotor recovery after spinal cord injury in male and female neonatal rats.

OBJECTIVE: It is not known how activation of the hypoxia-inducible factor (HIF) pathway in pericytes, cells of the microvascular wall, influences new capillary growth. We tested the hypothesis that HIF-activated pericytes promote angiogenesis in a neonatal model of spinal cord injury (SCI). METHODS: Human placental pericytes stimulated with cobalt chloride and naïve pericytes were injected into the site of a thoracic hemi-section of the spinal cord in rat pups on postnatal day three (P3). Hindlimb motor recovery and Doppler blood flow perfusion at the site of transection were measured on P10. Immunohistochemistry was used to visualize vessel and neurofilament density for quantification. RESULTS: Injection of HIF-activated pericytes resulted in greater vascular density in males but did not result in improved motor function for males or females. Injection of non-HIF-activated pericytes resulted improved motor function recovery in both sexes (males, 2.722 ± 0.31-fold score improvement; females, 3.824 ± 0.58-fold score improvement, P < .05) but produced no significant changes in vessel density. CONCLUSIONS: HIF-activated pericytes promote vascular density in males post-SCI. Acute delivery of non-HIF-activated pericytes at the site of injury can improve motor recovery post-SCI.

Acoustic Trauma Causes Cochlear Pericyte-to-Myofibroblast-Like Cell Transformation and Vascular Degeneration, and Transplantation of New Pericytes Prevents Vascular Atrophy.

Acoustic trauma disrupts cochlear blood flow and damages sensory hair cells. Damage and regression of capillaries after acoustic trauma have long been observed, but the underlying mechanism of pathology has not been understood. We show herein that loud sound causes change of phenotype from neural/glial antigen 2 positive/α-smooth muscle actin negative to neural/glial antigen 2 positive/α-smooth muscle actin positive in some pericytes (PCs) on strial capillaries that is strongly associated with up-regulation of transforming growth factor-ß1. The acoustic trauma also reduced capillary density and increased deposition of matrix proteins, particularly in the vicinity of transformed PCs. In a newly established in vitro three-dimensional endothelial cell (EC) and PC co-culture model, transformed PCs induced thicker capillary-like branches in ECs and increased collagen IV and laminin expression. Transplantation of exogenous PCs derived from neonatal day 10 mouse cochleae to acoustic traumatized cochleae, however, significantly attenuated the decreased vascular density in the stria. Transplantation of PCs pretransfected with adeno-associated virus 1-vascular endothelial growth factor-A165 under control of a hypoxia-response element markedly promotes vascular volume and blood flow, increased proliferation of PCs and ECs, and attenuated loud sound-caused loss in endocochlear potential and hearing. Our results indicate that loud sound-triggered PC transformation contributes to capillary wall thickening and regression, and young PC transplantation effectively rehabilitates the vascular regression and improves hearing.

Axon Degeneration Is Rescued with Human Umbilical Cord Perivascular Cells: A Potential Candidate for Neuroprotection After Traumatic Brain Injury.

Traumatic brain injury (TBI) leads to delayed secondary injury events consisting of cellular and molecular cascades that exacerbate the initial injury. Human umbilical cord perivascular cells (HUCPVCs) secrete neurotrophic and prosurvival factors. In this study, we examined the effects of HUCPVC in sympathetic axon and cortical axon survival models and sought to determine whether HUCPVC provide axonal survival cues. We then examined the effects of the HUCPVC in an in vivo fluid percussion injury model of TBI. Our data indicate that HUCPVCs express neurotrophic and neural survival factors. They also express and secrete relevant growth and survival proteins when cultured alone, or in the presence of injured axons. Coculture experiments indicate that HUCPVCs interact preferentially with axons when cocultured with sympathetic neurons and reduce axonal degeneration. Nerve growth factor withdrawal in axonal compartments resulted in 66 ± 3% axon degeneration, whereas HUCPVC coculture rescued axon degeneration to 35 ± 3%. Inhibition of Akt (LY294002) resulted in a significant increase in degeneration compared with HUCPVC cocultures (48 ± 7% degeneration). Under normoxic conditions, control cultures showed 39 ± 5% degeneration. Oxygen glucose deprivation (OGD) resulted in 58 ± 3% degeneration and OGD HUCPVC cocultures reduced degeneration to 34 ± 5% (p < 0.05). In an in vivo model of TBI, immunohistochemical analysis of NF200 showed improved axon morphology in HUCPVC-treated animals compared with injured animals. These data presented in this study indicate an important role for perivascular cells in protecting axons from injury and a potential cell-based therapy to treat secondary injury after TBI.

Non-fibro-adipogenic pericytes from human embryonic stem cells attenuate degeneration of the chronically injured mouse muscle.

Massive tears of the rotator cuff (RC) are associated with chronic muscle degeneration due to fibrosis, fatty infiltration, and muscle atrophy. The microenvironment of diseased muscle often impairs efficient engraftment and regenerative activity of transplanted myogenic precursors. Accumulating myofibroblasts and fat cells disrupt the muscle stem cell niche and myogenic cell signaling and deposit excess disorganized connective tissue. Therefore, restoration of the damaged stromal niche with non-fibro-adipogenic cells is a prerequisite to successful repair of an injured RC. We generated from human embryonic stem cells (hES) a potentially novel subset of PDGFR-ß+CD146+CD34-CD56- pericytes that lack expression of the fibro-adipogenic cell marker PDGFR-α. Accordingly, the PDGFR-ß+PDGFR-α- phenotype typified non-fibro-adipogenic, non-myogenic, pericyte-like derivatives that maintained non-fibro-adipogenic properties when transplanted into chronically injured murine RCs. Although administered hES pericytes inhibited developing fibrosis at early and late stages of progressive muscle degeneration, transplanted PDGFR-ß+PDGFR-α+ human muscle-derived fibro-adipogenic progenitors contributed to adipogenesis and greater fibrosis. Additionally, transplanted hES pericytes substantially attenuated muscle atrophy at all tested injection time points after injury. Coinciding with this observation, conditioned medium from cultured hES pericytes rescued atrophic myotubes in vitro. These findings imply that non-fibro-adipogenic hES pericytes recapitulate the myogenic stromal niche and may be used to improve cell-based treatments for chronic muscle disorders.

Early results of intra-articular micro-fragmented lipoaspirate treatment in patients with late stages knee osteoarthritis: a prospective study.

AIM: To analyze clinical and functional effects of intra-articular injection of autologous micro-fragmented lipoaspirate (MLA) in patients with late stage knee osteoarthritis (KOA). Secondary aims included classifying cell types contributing to the treatment effect, performing detailed MRI-based classification of KOA, and elucidating the predictors for functional outcomes. METHODS: This prospective, non-randomized study was conducted from June 2016 to February 2018 and enrolled 20 patients with late stage symptomatic KOA (Kellgren Lawrence grade III, n=4; and IV, n=16) who received an intra-articular injection of autologous MLA in the index knee joint. At baseline radiological KOA grade and MRI were assessed in order to classify the morphology of KOA changes. Stromal vascular fraction cells obtained from MLA samples were stained with antibodies specific for cell surface markers. Patients were evaluated at baseline and 12-months after treatment with visual analog scale (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and Knee Injury and Osteoarthritis Outcome Score (KOOS). RESULTS: Three patients (15%) received a total knee replacement and were not followed up completely. Seventeen patients (85%) showed a substantial pattern of KOOS and WOMAC improvement, significant in all accounts. KOOS score improved from 46 to 176% when compared with baseline, WOMAC decreased from 40 to 45%, while VAS rating decreased from 54% to 82% (all P values were <0.001). MLA contained endothelial progenitor cells, pericytes, and supra-adventitial adipose stromal cells as most abundant cell phenotypes. CONCLUSION: This study is among the first to show a positive effect of MLA on patients with late stages KOA.

Pericyte transplantation improves skeletal muscle recovery following hindlimb immobilization.

Conditions of extended bed rest and limb immobilization can initiate rapid and significant loss of skeletal muscle mass and function. Physical rehabilitation is standard practice following a period of disuse, yet mobility may be severely compromised, and recovery is commonly delayed or incomplete in special populations. Thus, a novel approach toward recovery of muscle mass is highly desired. Pericytes [neuron-glial antigen 2 (NG2)+CD31-CD45- (Lineage- [Lin-]) and CD146+Lin-] demonstrate capacity to facilitate muscle repair, yet the ability to enhance myofiber growth following disuse is unknown. In the current study, 3-4-mo-old mice were unilaterally immobilized for 14 d (IM) or immobilized for 14 d followed by 14 d of remobilization (RE). Flow cytometry and targeted gene expression analyses were completed to assess pericyte quantity and function following IM and RE. In addition, a transplantation study was conducted to assess the impact of pericytes on recovery. Results from targeted analyses suggest minimal impact of disuse on pericyte gene expression, yet NG2+Lin- pericyte quantity is reduced following IM (P < 0.05). Remarkably, pericyte transplantation recovered losses in myofiber cross-sectional area and the capillary-to-fiber ratio following RE, whereas deficits remained with vehicle alone (P = 0.01). These findings provide the first evidence that pericytes effectively rehabilitate skeletal muscle mass following disuse atrophy.-Munroe, M., Dvoretskiy, S., Lopez, A., Leong, J., Dyle, M. C., Kong, H., Adams, C. M., Boppart, M. D. Pericyte transplantation improves skeletal muscle recovery following hindlimb immobilization.

Role of TPBG (Trophoblast Glycoprotein) Antigen in Human Pericyte Migratory and Angiogenic Activity.

Objective- To determine the role of the oncofetal protein TPBG (trophoblast glycoprotein) in normal vascular function and reparative vascularization. Approach and Results- Immunohistochemistry of human veins was used to show TPBG expression in vascular smooth muscle cells and adventitial pericyte-like cells (APCs). ELISA, Western blot, immunocytochemistry, and proximity ligation assays evidenced a hypoxia-dependent upregulation of TPBG in APCs not found in vascular smooth muscle cells or endothelial cells. This involves the transcriptional modulator CITED2 (Atypical chemokine receptor 3 CBP/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich tail) and downstream activation of CXCL12 (chemokine [C-X-C motif] ligand-12) signaling through the CXCR7 (C-X-C chemokine receptor type 7) receptor and ERK1/2 (extracellular signal-regulated kinases 1/2). TPBG silencing by siRNA transfection downregulated CXCL12, CXCR7, and pERK (phospho Thr202/Tyr204 ERK1/2) and reduced the APC migratory and proangiogenic capacities. TPBG forced expression induced opposite effects, which were associated with the formation of CXCR7/CXCR4 (C-X-C chemokine receptor type 4) heterodimers and could be contrasted by CXCL12 and CXCR7 neutralization. In vivo Matrigel plug assays using APCs with or without TPBG silencing evidenced TPBG is essential for angiogenesis. Finally, in immunosuppressed mice with limb ischemia, intramuscular injection of TPBG-overexpressing APCs surpassed naïve APCs in enhancing perfusion recovery and reducing the rate of toe necrosis. Conclusions- TPBG orchestrates the migratory and angiogenic activities of pericytes through the activation of the CXCL12/CXCR7/pERK axis. This novel mechanism could be a relevant target for therapeutic improvement of reparative angiogenesis.

Pericytes as a Source of Osteogenic Cells in Bone Fracture Healing.

Pericytes are mesenchymal cells that surround the endothelial cells of small vessels in various organs. These cells express several markers, such as NG2, CD146, and PDGFRß, and play an important role in the stabilization and maturation of blood vessels. It was also recently revealed that like mesenchymal stem cells (MSCs), pericytes possess multilineage differentiation capacity, especially myogenic, adipogenic, and fibrogenic differentiation capacities. Although some previous studies have reported that pericytes also have osteogenic potential, the osteogenesis of pericytes can still be further elucidated. In the present study, we established novel methods for isolating and culturing primary murine pericytes. An immortalized pericyte line was also established. Multilineage induction of the pericyte line induced osteogenesis, adipogenesis, and chondrogenesis of the cells in vitro. In addition, pericytes that were injected into the fracture site of a bone fracture mouse model contributed to callus formation. Furthermore, in vivo pericyte-lineage-tracing studies demonstrated that endogenous pericytes also differentiate into osteoblasts and osteocytes and contribute to bone fracture healing as a cellular source of osteogenic cells. Pericytes can be a promising therapeutic candidate for treating bone fractures with a delayed union or nonunion as well as bone diseases causing bone defects.

Adipose-derived Human Perivascular Stem Cells May Improve Achilles Tendon Healing in Rats.

BACKGROUND: Achilles tendon rupture is a common injury and the best treatment option remains uncertain between surgical and nonoperative methods. Biologic approaches using multipotent stem cells such as perivascular stem cells pose a possible treatment option, although there is currently a paucity of evidence regarding their clinical therapeutic use. QUESTIONS/PURPOSES: The purpose of this study was to determine whether injected perivascular stem cells (PSCs) would (1) improve histologic signs of tendon healing (such as percent area of collagen); and (2) improve biomechanical properties (peak load or stiffness) in a rat model of Achilles tendon transection. METHODS: Two subtypes of PSCs were derived from human adipose tissue: pericytes (CD146CD34CD45CD31) and adventitial cells (CD146CD34CD45CD31). Thirty-two athymic rats underwent right Achilles transection and were randomized to receive injection with saline (eight tendons), hydrogel (four tendons), pericytes in hydrogel (four tendons), or adventitial cells in hydrogel (eight tendons) 3 days postoperatively with the left serving as an uninjured control. Additionally, a subset of pericytes was labeled with CM-diI to track cell viability and localization. At 3 weeks, the rats were euthanized, and investigators blinded to treatment group allocation evaluated tendon healing by peak load and stiffness using biomechanical testing and percent area of collagen using histologic analysis with picrosirius red staining. RESULTS: Histologic analysis showed a higher mean percent area collagen for pericytes (30%) and adventitial cells (28%) than hydrogel (21%) or saline (26%). However, a nonparametric statistical analysis yielded no statistical difference. Mechanical testing demonstrated that the pericyte group had a higher peak load than the saline group (41 ± 7 N versus 26 ± 9 N; mean difference 15 N; 95% confidence interval [CI], 4-27 N; p = 0.003) and a higher peak load than the hydrogel group (41 ± 7 N versus 25 ± 3 N; mean difference 16; 95% CI, 8-24 N; p = 0.001). The pericyte group demonstrated higher stiffness than the hydrogel group (36 ± 12 N/mm versus 17 ± 6 N/mm; mean difference 19 N/mm; 95% CI, 5-34 N/mm; p = 0.005). CONCLUSIONS: Our results suggest that injection of PSCs improves mechanical but not the histologic properties of early Achilles tendon healing. CLINICAL RELEVANCE: This is a preliminary study that provides more insight into the use of adipose-derived PSCs as a percutaneous therapy in the setting of Achilles tendon rupture. Further experiments to characterize the function of these cells may serve as a pathway to development of minimally invasive intervention aimed at improving nonoperative management while avoiding the complications associated with surgical treatment down the line.

The Stromal Vascular Fraction Improves Maintenance of the Fat Graft Volume: A Systematic Review.

BACKGROUND AND OBJECTIVES: With the continued application of fat grafting in plastic surgery, many studies have focused on various factors to improve maintenance of the fat graft volume, such as platelet-rich plasma, adipose-derived stromal/stem cells, and the stromal vascular fraction (SVF). In addition, many review articles have investigated the functions of platelet-rich plasma and adipose-derived stromal/stem cells in fat grafting, although the usefulness of the SVF remains unclear. The aim of the present review was to determine whether SVF use could maintain a fat graft. METHODS: A systematic review was conducted of the PubMed, Cochrane Central Register of Controlled Trials, and Embase databases of original articles published up to February 2018. RESULTS: Relevant articles were identified by screening the abstracts. A total of 58 full texts were initially identified. After exclusion, 17 articles, including 6 animal studies and 11 clinical studies, were included for analysis. CONCLUSIONS: Most studies found a significant and measurable long-term effect of SVF-enhanced fat grafting on breast augmentation and defects, wound healing, scaring, and facial aesthetic outcomes. Stromal vascular fraction use did not result in a higher instance of complications and, thus, can be considered a safe option for fat grafting.

Pericytes reduce inflammation and collagen deposition in acute wounds.

BACKGROUND: Pericytes have been shown to have mesenchymal stromal cell-like properties and play a role in tissue regeneration. The goal of this study was to determine whether the addition of a pericyte sheet to a full-thickness dermal wound would enhance the healing of an acute wound. METHODS: Human muscle-derived pericytes and human dermal fibroblasts were formed into cell sheets, then applied to full-thickness excisional wounds on the dorsum of nu/nu mice. Histology was performed to evaluate epidermal and dermal reformation, inflammation and fibrosis. In addition, real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to determine cytokine response. RESULTS: Pericytes were detected in the wounds until day 16 but not fibroblasts. Decrease in wound size was noted in pericyte sheet-treated wounds. Enhanced neo-vascularization and healthy granulation tissue formation were noted in the pericyte-treated wounds. Expression of type I collagen messenger RNA (mRNA) was significantly higher in the fibroblast-treated group, whereas Type III collagen mRNA showed significant increase in the pericyte group at days 3, 6 and 9 compared with the fibroblast and no-cell groups. Trichrome staining revealed thick unorganized collagen fibrils in the fibroblast-treated wounds, whereas pericyte-treated wounds contained thinner and more alligned collagen fibrils. Tumor necrosis factor (TNF)-α mRNA levels were increased in the fibroblast-treated wounds compared with pericyte-treated wounds. DISCUSSION: The addition of pericytes may confer beneficial effects to wound healing resulting in reduced recruitment of inflammatory cells and collagen I deposition, potential to enhance wound closure and better collagen alignment promoting stronger tissue.

Type 2 diabetes impairs the ability of skeletal muscle pericytes to augment postischemic neovascularization in db/db mice.

Peripheral artery disease is an atherosclerotic occlusive disease that causes limb ischemia and has few effective noninterventional treatments. Stem cell therapy is promising, but concomitant diabetes may limit its effectiveness. We evaluated the therapeutic potential of skeletal muscle pericytes to augment postischemic neovascularization in wild-type and type 2 diabetic (T2DM) mice. Wild-type C57BL/6J and leptin receptor spontaneous mutation db/db T2DM mice underwent unilateral femoral artery excision to induce limb ischemia. Twenty-four hours after ischemia induction, CD45-CD34-CD146+ skeletal muscle pericytes or vehicle controls were transplanted into ischemic hindlimb muscles. At postoperative day 28, pericyte transplantation augmented blood flow recovery in wild-type mice (79.3 ± 5% vs. 61.9 ± 5%; P = 0.04), but not in T2DM mice (48.6% vs. 46.3 ± 5%; P = 0.51). Pericyte transplantation augmented collateral artery enlargement in wild-type (26.7 ± 2 µm vs. 22.3 ± 1 µm, P = 0.03), but not T2DM mice (20.4 ± 1.4 µm vs. 18.5 ± 1.2 µm, P = 0.14). Pericyte incorporation into collateral arteries was higher in wild-type than in T2DM mice ( P = 0.002). Unexpectedly, pericytes differentiated into Schwann cells in vivo. In vitro, Insulin increased Nox2 expression and decreased tubular formation capacity in human pericytes. These insulin-induced effects were reversed by N-acetylcysteine antioxidant treatment. In conclusion, T2DM impairs the ability of pericytes to augment neovascularization via decreased collateral artery enlargement and impaired engraftment into collateral arteries, potentially via hyperinsulinemia-induced oxidant stress. While pericytes show promise as a unique form of stem cell therapy to increase postischemic neovascularization, characterizing the molecular mechanisms by which T2DM impairs their function is essential to achieve their therapeutic potential.

Transplantation of Allogeneic Pericytes Improves Myocardial Vascularization and Reduces Interstitial Fibrosis in a Swine Model of Reperfused Acute Myocardial Infarction.

BACKGROUND: Transplantation of adventitial pericytes (APCs) promotes cardiac repair in murine models of myocardial infarction. The aim of present study was to confirm the benefit of APC therapy in a large animal model. METHODS AND RESULTS: We performed a blind, randomized, placebo-controlled APC therapy trial in a swine model of reperfused myocardial infarction. A first study used human APCs (hAPCs) from patients undergoing coronary artery bypass graft surgery. A second study used allogeneic swine APCs (sAPCs). Primary end points were (1) ejection fraction as assessed by cardiac magnetic resonance imaging and (2) myocardial vascularization and fibrosis as determined by immunohistochemistry. Transplantation of hAPCs reduced fibrosis but failed to improve the other efficacy end points. Incompatibility of the xenogeneic model was suggested by the occurrence of a cytotoxic response following in vitro challenge of hAPCs with swine spleen lymphocytes and the failure to retrieve hAPCs in transplanted hearts. We next considered sAPCs as an alternative. Flow cytometry, immunocytochemistry, and functional/cytotoxic assays indicate that sAPCs are a surrogate of hAPCs. Transplantation of allogeneic sAPCs benefited capillary density and fibrosis but did not improve cardiac magnetic resonance imaging indices of contractility. Transplanted cells were detected in the border zone. CONCLUSIONS: Immunologic barriers limit the applicability of a xenogeneic swine model to assess hAPC efficacy. On the other hand, we newly show that transplantation of allogeneic sAPCs is feasible, safe, and immunologically acceptable. The approach induces proangiogenic and antifibrotic benefits, though these effects were not enough to result in functional improvements.