Paula K. Shireman

Reproducibility of quantitative RT-PCR array in miRNA expression profiling and comparison with microarray analysis

BackgroundMicroRNAs (miRNAs) have critical functions in various biological processes. MiRNA profiling is an important tool for the identification of differentially expressed miRNAs in normal cellular and disease processes. A technical challenge remains for high-throughput miRNA expression analysis as the number of miRNAs continues to increase with in silico prediction and experimental verification. Our study critically evaluated the performance of a novel miRNA expression profiling approach, quantitative RT-PCR array (qPCR-array), compared to miRNA detection with oligonucleotide microchip (microarray).ResultsHigh reproducibility with qPCR-array was demonstrated by comparing replicate results from the same RNA sample. Pre-amplification of the miRNA cDNA improved sensitivity of the qPCR-array and increased the number of detectable miRNAs. Furthermore, the relative expression levels of miRNAs were maintained after pre-amplification. When the performance of qPCR-array and microarrays were compared using different aliquots of the same RNA, a low correlation between the two methods (r = -0.443) indicated considerable variability between the two assay platforms. Higher variation between replicates was observed in miRNAs with low expression in both assays. Finally, a higher false positive rate of differential miRNA expression was observed using the microarray compared to the qPCR-array.ConclusionOur studies demonstrated high reproducibility of TaqMan qPCR-array. Comparison between different reverse transcription reactions and qPCR-arrays performed on different days indicated that reverse transcription reactions did not introduce significant variation in the results. The use of cDNA pre-amplification increased the sensitivity of miRNA detection. Although there was variability associated with pre-amplification in low abundance miRNAs, the latter did not involve any systemic bias in the estimation of miRNA expression. Comparison between microarray and qPCR-array indicated superior sensitivity and specificity of qPCR-array.

MCP-1 deficiency causes altered inflammation with impaired skeletal muscle regeneration

We examined the role of MCP‐1, a potent chemotactic and activating factor for macrophages, in perfusion, inflammation, and skeletal muscle regeneration post‐ischemic injury. MCP‐1−/− or C57Bl/6J control mice [wild‐type (WT)] underwent femoral artery excision (FAE). Muscles were collected for histology, assessment of tissue chemokines, and activity measurements of lactate dehydrogenase (LDH) and myeloperoxidase. In MCP‐1−/− mice, restoration of perfusion was delayed, and LDH and fiber size, indicators of muscle regeneration, were decreased. Altered inflammation was observed with increased neutrophil accumulation in MCP‐1−/− versus WT mice at Days 1 and 3 (P≤0.003), whereas fewer macrophages were present in MCP‐1−/− mice at Day 3. As necrotic tissue was removed in WT mice, macrophages decreased (Day 7). In contrast, macrophage accumulation in MCP‐1−/− was increased in association with residual necrotic tissue and impaired muscle regeneration. Consistent with altered inflammation, neutrophil chemotactic factors (keratinocyte‐derived chemokine and macrophage inflammatory protein‐2) were increased at Day 1 post‐FAE. The macrophage chemotactic factor MCP‐5 was increased significantly in WT mice at Day 3 compared with MCP‐1−/− mice. However, at post‐FAE Day 7, MCP‐5 was significantly elevated in MCP‐1−/− mice versus WT mice. Addition of exogenous MCP‐1 did not induce proliferation in murine myoblasts (C2C12 cells) in vitro. MCP‐1 is essential for reperfusion and the successful completion of normal skeletal muscle regeneration after ischemic tissue injury. Impaired muscle regeneration in MCP‐1−/− mice suggests an important role for macrophages and MCP‐1 in tissue reparative processes.

Chemokines and diabetic wound healing

Chemokines are critical for white blood cell recruitment to injured tissues and play an important role in normal wound healing processes. In contrast, impaired wound healing in diabetic patients is accompanied by decreased early inflammatory cell infiltration but persistence of neutrophils and macrophages in the chronic, nonhealing wounds. These changes in inflammatory cell recruitment occur in conjunction with alterations in chemokine and growth factor expression. In addition to leukocyte trafficking, many different cell types, including endothelial cells, fibroblasts, and keratinocytes, produce and respond to chemokines, and these interactions are altered in diabetic wounds. Thus, the chemokine system may have both direct and inflammatory-mediated effects on many different aspects of diabetic wound healing. The potential roles of chemokines and inflammatory or immune cells in nonhealing diabetic wounds, including impairments in growth factor expression, angiogenesis, extracellular matrix formation, and reepithelialization, are examined.

Bone marrow-derived cell regulation of skeletal muscle regeneration

Limb regeneration requires the coordination of multiple stem cell populations to recapitulate the process of tissue formation. Therefore, bone marrow (BM) ‐derived cell regulation of skeletal muscle regeneration was examined in mice lacking the CC chemokine receptor 2 (CCR2). Myofiber size, numbers of myogenic progenitor cells (MPCs), and recruitment of BM‐derived cells and macrophages were assessed after cardiotoxin‐induced injury of chimeric mice produced by transplanting BM from wild‐type (WT) or CCR2−/− mice into irradiated WT or CCR2−/− host mice. Regardless of the host genotype, muscle regeneration and recruitment of BM‐derived cells and macrophages were similar in mice replenished with WT BM, whereas BM‐derived cells and macrophage accumulation were decreased and muscle regeneration was impaired in all animals receiving CCR2−/− BM. Furthermore, numbers of MPCs (CD34+/Sca‐1−/CD45− cells) were significantly increased in mice receiving CCR2−/− BM despite the decreased size of regenerated myofibers. Thus, the expression of CCR2 on BM‐derived cells regulated macrophage recruitment into injured muscle, numbers of MPC, and the extent of regenerated myofiber size, all of which were independent of CCR2 expression on host‐derived cells. Future studies in regenerative medicine must include consideration of the role of BM‐derived cells, possibly macrophages, in CCR2‐dependent events that regulate effective skeletal muscle regeneration.—Sun, D., Martinez, C. O., Ochoa, O., Ruiz‐Willhite, L., Bonilla, J. R, Centonze, V. E., Waite, L. L., Michalek, J. E., McManus, L. M., Shireman, P. K. Bone marrow‐derived cell regulation of skeletal muscle regeneration. FASEB J. 23, 382–395 (2009)

Regulation of skeletal muscle regeneration by CCR2-activating chemokines is directly related to macrophage recruitment

Muscle regeneration requires CC chemokine receptor 2 (CCR2) expression on bone marrow-derived cells; macrophages are a prominent CCR2-expressing cell in this process. CCR2-/- mice have severe impairments in angiogenesis, macrophage recruitment, and skeletal muscle regeneration following cardiotoxin (CTX)-induced injury. However, multiple chemokines activate CCR2, including monocyte chemotactic proteins (MCP)-1, -3, and -5. We hypothesized that MCP-1 is the chemokine ligand that mediates the impairments present in CCR2-/- mice. We examined muscle regeneration, capillary density, and cellular recruitment in MCP-1-/- and CCR2-/- mice following injury. Muscle regeneration and adipocyte accumulation, but not capillary density, were significantly impaired in MCP-1-/- compared with wild-type (WT) mice; however, muscle regeneration and adipocyte accumulation impairments were not as severe as observed in CCR2-/- mice. Although tissue levels of MCP-5 were elevated in MCP-1-/- mice compared with WT, the administration of MCP-5 neutralizing antibody did not alter muscle regeneration in MCP-1-/- mice. While neutrophil accumulation after injury was similar in all three mouse strains, macrophage recruitment was highest in WT mice, intermediate in MCP-1-/- mice, and severely impaired in CCR2-/- mice. In conclusion, while the absence of MCP-1 resulted in impaired macrophage recruitment and muscle regeneration, MCP-1-/- mice exhibit an intermediate phenotype compared with CCR2-/- mice. Intermediate macrophage recruitment in MCP-1-/- mice was associated with similar capillary density to WT, suggesting that fewer macrophages may be needed to restore angiogenesis vs. muscle regeneration. Finally, other chemokines, in addition to MCP-1 and MCP-5, may activate CCR2-dependent regenerative processes resulting in an intermediate phenotype in MCP-1-/- mice.

Altered Macrophage Phenotype Transition Impairs Skeletal Muscle Regeneration

Monocyte/macrophage polarization in skeletal muscle regeneration is ill defined. We used CD11b-diphtheria toxin receptor transgenic mice to transiently deplete monocytes/macrophages at multiple stages before and after muscle injury induced by cardiotoxin. Fat accumulation within regenerated muscle was maximal when ablation occurred at the same time as cardiotoxin-induced injury. Early ablation (day 1 after cardiotoxin) resulted in the smallest regenerated myofiber size together with increased residual necrotic myofibers and fat accumulation. However, muscle regeneration after late (day 4) ablation was similar to controls. Levels of inflammatory cells in injured muscle following early ablation and associated with impaired muscle regeneration were determined by flow cytometry. Delayed, but exaggerated, monocyte [CD11b(+)(CD90/B220/CD49b/NK1.1/Ly6G)(-)(F4/80/I-Ab/CD11c)(-)Ly6C(+/-)] accumulation occurred; interestingly, Ly6C(+) and Ly6C(-) monocytes were present concurrently in ablated animals and control mice. In addition to monocytes, proinflammatory, Ly6C(+) macrophage accumulation following early ablation was delayed compared to controls. In both groups, CD11b(+)F4/80(+) cells exhibited minimal expression of the M2 markers CD206 and CD301. Nevertheless, early ablation delayed and decreased the transient accumulation of CD11b(+)F4/80(+)Ly6C(-)CD301(-) macrophages; in control animals, the later tissue accumulation of these cells appeared to correspond to that of anti-inflammatory macrophages, determined by cytokine production and arginase activity. In summary, impairments in muscle regeneration were associated with exaggerated monocyte recruitment and reduced Ly6C(-) macrophages; the switch of macrophage/monocyte subsets is critical to muscle regeneration.

Surgical management of atheroembolization

PURPOSE Atheroembolization may cause limb loss or organ failure. Surgical outcome data are limited. We report the largest series of atheroembolization focusing on patterns of disease, surgical treatment and outcome. METHODS One hundred patients (70 men), mean age 62 +/- 11 years, operated on for lower extremity, visceral, or nonthoracic outlet upper extremity atheroemboli were identified prospectively and monitored over a 12-year period. The atheroembolic source was localized by use of a combination of computed tomography scanning (n = 55), arteriography (n = 93), duplex scanning (n = 25), transesophageal echocardiography (n = 6), and magnetic resonance imaging (n = 4). Occlusive aortoiliac disease (47 patients) and small aortic aneurysms (20 patients; mean aneurysm size 3.5 +/- 0.8 cm) were the most common source of atheroemboli. Imaging studies revealed 12 patients with extensive suprarenal aortic thrombus. Correction of the embolic source was achieved with aortic bypass (n = 52), aortoiliac endarterectomy and patch (n = 11), femoral or popliteal endarterectomy and patch (n = 11), infrainguinal bypass (n = 3), extraanatomic reconstruction (n = 6), graft revision (n = 3), upper extremity bypass (n = 11), or upper extremity endarterectomy and patch (n = 3). RESULTS All four deaths within 30 days and all seven deaths within the first 6 months after operation were among the 12 patients with suprarenal aortic thrombus. The cumulative survival probabilities for all patients at 1, 3, and 5 years were 89%, 83%, and 73%, respectively. After operation, nine patients required major leg amputations and 10 required toe amputations. Renal atheroemboli led to hemodialysis in 10 patients. Recurrent embolic events occurred in five of 97 patients monitored for a mean of 32 months. All five recurrences occurred in the first 8 months after operation. Three patients with recurrent emboli had suprarenal aortic disease, one of whom had undergone axillofemorofemoral bypass. Four of 15 patients receiving postoperative warfarin anticoagulation had development of recurrent embolism. Only one patient not receiving postoperative warfarin had a recurrent event (p < 0.05 by Fisher exact test). CONCLUSION The atheroembolic source is the aorta or iliac arteries in two thirds of patients who underwent operation. Computed tomography scanning of the aorta is a useful diagnostic technique. The source of the emboli can be eliminated surgically with low mortality or limb loss rates except when the suprarenal aorta is involved.

Temporal microRNA expression during in vitro myogenic progenitor cell proliferation and differentiation: regulation of proliferation by miR-682

MicroRNAs (miRNAs) regulate gene expression by repressing target genes at the posttranscriptional level. Since miRNAs have unique expression profiles in different tissues, they provide pivotal regulation of many biological processes. The present study defined miRNA expression during murine myogenic progenitor cell (MPC) proliferation and differentiation to identify miRNAs involved in muscle regeneration. Muscle-related gene expression analyses revealed that the time course and expression of myosin heavy chain (MHC) and transcription factors (Myf5, MyoD, myogenin, and Pax7) were similar during in vitro MPC proliferation/differentiation and in vivo muscle regeneration. Comprehensive profiling revealed that 139 or 16 miRNAs were significantly changed more than twofold [false discovery rate (FDR) < 0.05] during MPC differentiation or proliferation, respectively; cluster analyses revealed five distinct patterns of miRNA expression during the time course of MPC differentiation. Not unexpectedly, the largest miRNA changes occurred in muscle-specific miRNAs (miR-1, -133a, and -499), which were upregulated >10-fold during MPC differentiation (FDR < 0.01). However, several previously unreported miRNAs were differentially expressed, including miR-10b, -335-3p, and -682. Interestingly, the temporal patterns of miR-1, -499, and -682 expression during in vitro MPC proliferation/differentiation were remarkably similar to those observed during in vivo muscle regeneration. Moreover, in vitro inhibition of miR-682, the only miRNA upregulated in proliferating compared with quiescent MPC, led to decreased MPC proliferation, further validating our in vitro assay system for the identification of miRNAs involved in muscle regeneration. Thus the differentially expressed miRNAs identified in the present study could represent new regulatory elements in MPC proliferation and differentiation.

The S130K fibroblast growth factor–1 mutant induces heparin-independent proliferation and is resistant to thrombin degradation in fibrin glue

OBJECTIVE Site-directed mutagenesis is an important technique that can alter cytokine function, thereby eliciting desired responses. S130K is a mutation of fibroblast growth factor-1 (FGF-1), with lysine replacing serine in the heparin-binding site. We measured molecular stability and mitogenic activity of FGF-1 and S130K, both in the media and when suspended in fibrin glue (FG), on smooth muscle cells (SMCs) and endothelial cells (ECs) to determine if the mutation altered the function and potential clinical applicability. METHODS EC and SMC proliferation of soluble FGF-1 or S130K at 0, 0. 1, 1, 10, or 100 ng/mL with heparin at 0, 5, 50, or 500 units (U)/mL was measured on growth-arrested cells in serum-free media. EC and SMC proliferation assays with cells on FG containing either FGF-1 or S130K at 0, 1, 10, 100, or 1000 ng/mL in combination with heparin at 0, 5, 50 or 500 U/mL were also performed during the exponential growth phase. Molecular degradation by thrombin was measured by sodium dodecylsulfate-polyacrylamide gel electrophoresis. RESULTS S130K induces greater EC and SMC proliferation in the absence of heparin than FGF-1 does (P <.0001 for both the 10 and 100 ng/mL doses). S130K is also significantly more potent than FGF-1 in the presence of heparin. Heparin in the media enhances cytokine-induced SMC and EC proliferation at doses of 5 U/mL, but inhibits SMC proliferation at concentrations of 500 U/mL. For the FG data, unlike FGF-1, S130K induces EC and SMC proliferation in the absence of heparin. The addition of 5 U/mL of heparin enhances the proliferation induced by S130K. For ECs, as the heparin dose increases to 50 U/mL, proliferation decreases, as compared with the 5 U/mL concentration when either FGF-1 or S130K in the FG was compared at concentrations of 10, 100, and 1000 ng/mL (P <.01). S130K is more potent in FG than is FGF-1 both with and without heparin and exhibits maximal EC and SMC proliferation at 10 ng/mL, whereas FGF-1 activity is maximal at 100 ng/mL. Gel electrophoresis demonstrated that S130K was relatively more resistant to thrombin degradation than FGF-1. CONCLUSIONS Site-directed mutagenesis changed the potency and the heparin dependency on cellular proliferation of FGF-1 in vitro. These techniques should allow the delivery of mutant growth factors to areas of vascular intervention to induce specific, desired responses. We believe that these studies will enhance our knowledge of the function of various regions of the FGF-1 molecule, allowing us to more precisely design increasingly more useful FGF-1 mutants.

MiR-351 transiently increases during muscle regeneration and promotes progenitor cell proliferation and survival upon differentiation

MicroRNAs (miRNAs) regulate many biological processes including muscle development. However, little is known regarding miRNA regulation of muscle regeneration. Murine tibialis anterior muscle was evaluated after cardiotoxin-induced injury and used for global miRNA expression analysis. From day 1 through day 21 following injury, 298 miRNAs were significantly changed at least at one time point, including 86 miRNAs that were altered >10-fold compared with uninjured skeletal muscle. Temporal miRNA expression patterns included inflammation-related miRNAs (miR-223 and -147) that increased immediately after injury; this pattern contrasted to that of mature muscle-specific miRNAs (miR-1, -133a, and -499) that abruptly decreased following injury followed by upregulation in later regenerative events. Another cluster of miRNAs were transiently increased in the early days of muscle regeneration including miR-351, a miRNA that was also transiently expressed during myogenic progenitor cell (MPC) differentiation in vitro. Based on computational predictions, further studies demonstrated that E2f3 was a target of miR-351 in myoblasts. Moreover, knockdown of miR-351 expression inhibited MPC proliferation and promoted apoptosis during MPC differentiation, whereas miR-351 overexpression protected MPC from apoptosis during differentiation. Collectively, these observations suggest that miR-351 is involved in both the maintenance of MPC proliferation and the transition into differentiated myotubes. Thus, a novel, time-dependent sequence of molecular events during muscle regeneration has been identified; miR-351 inhibits E2f3 expression, a key regulator of cell cycle progression and proliferation, and promotes MPC proliferation and protects early differentiating MPC from apoptosis, important events in the hostile tissue environment after acute muscle injury.