Pericytes are multipotent stem cells located in the perivascular space throughout the microvasculature. Their ability to differentiate into mesodermal cell types and modulate the local tissue environment through paracrine signaling makes pericytes a novel therapeutic target, but their prospective isolation and identification preclude their widespread use in therapeutic research. PURPOSE: To use novel flow cytometry methods to isolate and characterize pericytes from murine skeletal muscle. METHODS: Gastrocnemius and soleus complexes were dissected from CD1 mice, aged 3 months. Muscles were digested using collagenase into a single cell suspension. Cells were filtered through 70 um and 40 um mesh then resuspended in staining medium. Cells were stained at 4°C for 20 minutes with the following antibodies: CD45-BB515 (1:400), CD34-BV421 (1:100), CD56-PE (1:100) and CD146-Alexa647 (1:100). Fluorescence activated cell sorting (FACS) was used to sort CD45-CD56-CD34-CD146+ pericytes using appropriate unstained, single stained, and fluorescence minus one controls. P0 cells were plated on 0.2% gelatin coated plates in EBM-2 complete medium. At 70% confluence, P0 cells were split 1:1 on polystyrene tissue culture plates, and split 1:2 thereafter. Flow cytometry was used to determine the expression of pericyte markers (NG2 and PDGFβ), mesenchymal cell markers (CD73, CD90, and CD105), endothelial (CD31) and hematopoietic cells (CD45) markers on P5 cells. Characterization data were analyzed for the percentage of positive cells and median fluorescent intensity relative to unstained control (nMFI). RESULTS: Pericytes were positive for the pericyte marker CD146 (15.5%, nMFI 102.4), weakly positive for PDGFRβ (10.8%, nMFI 7.5), and weakly positive for NG2 (7.0%, nMFI 6.9). Cultured pericytes were positive for mesenchymal stem cell markers CD73 (52.9%, nMFI 56.9), and CD105 (35.3%, nMFI 9.5), but negative for CD90 (4.9%, nMFI 6.3). Pericytes were negative for the endothelial marker CD31 (2.1%, nMFI 4.4) and hematopoietic marker CD45 (5.4%, nMFI 7.9). CONCLUSIONS: FACS sorting for CD45-CD56-CD34-CD146+ cells yields murine pericytes that can be reliably isolated, definitively characterized, and may be suitable for in vivo and in vitro experiments to test the efficacy of pericyte cell therapy for various diseases.

Pericytes are skeletal muscle resident, multipotent stem cells that are localized to the microvasculature. In vivo, studies have shown that they respond to damage through activation of nuclear-factor kappa-B (NF-κB), but the downstream effects of NF-κB activation on endothelial cell proliferation and cell-cell signaling during repair remain unknown. The purpose of this study was to examine pericyte NF-κB activation in a model of skeletal muscle damage; and use genetic manipulation to study the effects of changes in pericyte NF-κB activation on endothelial cell proliferation and cytokine secretion. We utilized scratch injury to C2C12 cells in coculture with human primary pericytes to assess NF-κB activation and monocyte chemoattractant protein-1 (MCP-1) secretion from pericytes and C2C12 cells. We also cocultured endothelial cells with pericytes that expressed genetically altered NF-κB activation levels, and then quantified endothelial cell proliferation and screened the conditioned media for secreted cytokines. Pericytes trended toward greater NF-κB activation in injured compared to control cocultures (P = 0.085) and in comparison to C2C12 cells (P = 0.079). Second, increased NF-κB activation in pericytes enhanced the proliferation of cocultured endothelial cells (1.3-fold, P = 0.002). Finally, we identified inflammatory signaling molecules, including MCP-1 and interleukin 8 (IL-8) that may mediate the crosstalk between pericytes and endothelial cells. The results of this study show that pericyte NF-κB activation may be an important mechanism in skeletal muscle repair with implications for the development of therapies for musculoskeletal and vascular diseases, including peripheral artery disease.

This study aimed to evaluate an in vitro model of cross talk between pericytes and skeletal muscle cells following scrape injury. Nuclear factor‐kappa B (NF‐κB) and monocyte chemoattractant protein‐1 (MCP‐1) are important in the in vivo response to muscle stress and were chosen to evaluate the model. C2C12 cells were co‐cultured with human primary pericytes (HPPs) in transwell inserts. Nuclear NF‐κB/p65 DNA binding activity and MCP‐1 concentration were quantified via ELISA at baseline (BSLN), 3h, 6h, and 24h following scrape injury (INJ) to C2C12 cells. In C2C12 cells, p65 DNA binding activity was significantly elevated at 3h (2.5 fold, p=0.027) and 24h (3.57 fold, p=0.001), relative to BSLN, with no effect of INJ (p=0.698) relative to CON. In HPPs, p65 DNA binding activity was increased relative to BSLN at 6h (2.0 fold, p=0.007), and 24h (2.33 fold, p=0.001). HPPs trended towards greater p65 DNA binding activity in INJ compared to CON (p=0.085) and in comparison to C2C12 cells (p=0.079). At 24h, HPP MCP‐1 secretion was first detected and exceeded C2C12 MCP‐1 secretion (2.1 fold, p<0.001). These data reveal 1) this model is useful for evaluating secreted proteins involved in pericyte‐skeletal muscle cross talk, which are mostly unknown at this time, and 2) novel information regarding HPP MCP‐1 secretion in response to C2C12 scrape injury. This research was supported by an ACSM Foundation Doctoral Student Research Grant.

PURPOSE:This study compared endurance and neuromuscular function after bouts of low-load (LL), high-load (HL), and LL blood flow-restricted (LL(BFR)) resistance exercise. METHODS:Eight recreationally active male subjects completed three sets of dynamic knee extensions to volitional failure under three conditions: HL (70% peak torque), LL (20% peak torque), and LL(BFR) (20% peak torque with an occlusive cuff inflated to 180 mm Hg wrapped around the thigh). Before and immediately after exercise, isometric torque, central activation, electrically evoked torque, and muscle activation via surface EMG were measured. RESULTS:Isometric torque and evoked torque decreased an average of 37% and 40%, respectively (P < 0.01) in all conditions after exercise. There were no differences in the toque decrements between the conditions (P > 0.05). Percent central activation did not change after any condition (P = 0.09). Rate of torque development declined an average of 26% after all three conditions (P = 0.003), and rate of half-relaxation time was depressed by 48% after the HL condition (P = 0.004) only. EMG amplitude was greater in the HL condition at the beginning and end of exercise compared with the LL and LL(BFR) conditions (P = 0.001). At the end of exercise, EMG amplitude rose 19% (P = 0.02) and was not different among conditions (P > 0.05). Subjects performed more repetitions during the LL and LL(BFR) conditions (P < 0.05). CONCLUSION:Although LL and LL(BFR) resistance exercise to volitional failure exhibit lower levels of muscle activation than HL exercise, similar torque decrements occur after all bouts of resistance exercise, and the muscle fatigue can be attributed to peripheral factors.

Resistance training at low loads with a blood flow restriction has been shown to be as fatiguing as traditional high-load exercise and may provide an alternative for individuals who cannot participate in high load resistance training. Evaluating the origins of muscle fatigue may provide further insight into the mechanisms involved in blood flow restricted resistance exercise. PURPOSE: To compare the acute effects of low load exercise (LL), high load exercise (HL), and low load exercise with a blood flow restriction (LLBFR) fatigue protocols on neuromuscular function. METHODS: Eight subjects (8 recreationally active males, age 22±2.2y, height 177.6 ± 3.9 cm, weight 71.8 ± 17.4kg) completed 3 exercise conditions: HL (70% of peak torque), LL (20% of peak torque), and LLBFR (20% of peak torque with an occlusive cuff inflated to 180 mmHg wrapped proximally around the thigh for the duration of the exercise). During each condition, subjects performed 3 sets of knee extensions to volitional failure. Central activation, maximal voluntary contraction (MVC) force and training volume were assessed before and after each condition. RESULTS: Exercise volume (load X repetitions) was similar across all 3 conditions (HL: 4156 ± 1327; LL: 9070 ± 6603; LLBFR: 6018 ± 3280 Nm, P=0.08), MVC force decreased significantly (P<0.01) after each exercise condition, but did not differ across conditions (HL:-31.8 ± 9.0; LL: -40.8 ± 17.1; LLBFR: -38.1 ± 15.3%; P=0.35). Central activation did not change significantly after the exercise protocol (P=0.38) and this was similar between the exercise conditions (P=0.18). CONCLUSIONS: Significant decreases in MVC force over time, but not across conditions, suggests that the chosen protocols were equally fatiguing at their similar volumes. Decrements in central activation were not evident suggesting that fatigue may occur at the muscular level during these exercise protocols.