Oscar Ochoa

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.

Abdominal wall stability and flap complications after deep inferior epigastric perforator flap breast reconstruction: does body mass index make a difference? Analysis of 418 patients and 639 flaps.

Background: Promoted by reports of decreased donor-site morbidity, deep inferior epigastric perforator (DIEP) flaps have gained significant popularity. Increasing body mass index is associated with poor outcomes in breast reconstruction using traditional techniques. The authors aimed to define complications with increasing body mass index among patients undergoing DIEP flap breast reconstruction. Methods: A retrospective analysis of 639 DIEP flaps in 418 patients was performed. Patients were stratified into five groups based on body mass index. Data regarding medical comorbidities, adjuvant therapies, timing of reconstruction, active tobacco use, and surgical history were collected. Primary outcomes were compared among groups. Results: The average body mass index for the entire population was 28.3 (range, 17 to 42). Increasing body mass index was associated with increased incidence of hypertension, previous abdominal operations, and length of follow-up. Flap complications stratified by group demonstrated significantly increased delayed wound healing complications in severely obese patients compared with lower body mass index groups. Donor-site complications stratified by body mass index demonstrated significantly increased delayed wound healing and overall complications among morbidly obese patients compared with other groups. Incidence of abdominal wall bulging and hernia formation was not significantly different among groups. Conclusions: Increasing body mass index predisposes patients to delayed wound healing complications in both flap and donor-site locations. Nevertheless, overall flap complications remain similar across all body mass index groups. Abdominal wall stability was maintained. Given a similar flap complication profile and maintenance of abdominal stability, DIEP flaps are recommended in patients with increased body mass index. CLINICAL QUESTION/LEVEL OF EVDENCE: Risk, II.

Attenuation of RNA polymerase II pausing mitigates BRCA1-associated R-loop accumulation and tumorigenesis

Most BRCA1-associated breast tumours are basal-like yet originate from luminal progenitors. BRCA1 is best known for its functions in double-strand break repair and resolution of DNA replication stress. However, it is unclear whether loss of these ubiquitously important functions fully explains the cell lineage-specific tumorigenesis. In vitro studies implicate BRCA1 in elimination of R-loops, DNA-RNA hybrid structures involved in transcription and genetic instability. Here we show that R-loops accumulate preferentially in breast luminal epithelial cells, not in basal epithelial or stromal cells, of BRCA1 mutation carriers. Furthermore, R-loops are enriched at the 5′ end of those genes with promoter-proximal RNA polymerase II (Pol II) pausing. Genetic ablation of Cobra1, which encodes a Pol II-pausing and BRCA1-binding protein, ameliorates R-loop accumulation and reduces tumorigenesis in Brca1-knockout mouse mammary epithelium. Our studies show that Pol II pausing is an important contributor to BRCA1-associated R-loop accumulation and breast cancer development.

Salvage of intraoperative deep inferior epigastric perforator flap venous congestion with augmentation of venous outflow: flap morbidity and review of the literature.

Background: Breast reconstruction with deep inferior epigastric perforator (DIEP) flaps has gained considerable popularity due to reduced donor-site morbidity. Previous studies have identified the superficial venous system as the dominant outflow to DIEP flaps. DIEP flap venous congestion occurs if superficial venous outflow via the deep venous system is insufficient for effective flap drainage. Although augmentation of venous outflow through a second venous anastomosis may relieve venous congestion, effects on flap morbidity remain ill defined. Methods: A retrospective analysis of 1616 patients who underwent 2618 DIEP flap breast reconstructions between March 2005 and January 2012 was performed. Patients with intraoperative venous congestion underwent a second venous anastomosis. Preoperative demographic data and methods used to relieve venous congestion were recorded. Incidence of flap morbidity was calculated and compared with a group of 418 controls having 639 DIEP flap breast reconstructions with no venous congestion. Results: Venous augmentation was required to relieve venous congestion in 87 (3.3%) DIEP flaps on 81 patients. The superficial inferior epigastric vein or accompanying deep inferior epigastric venae comitantes was used to augment venous outflow. Preoperative comorbidities were similar between both groups. Patients requiring a second venous anastomosis had a longer operative time and length of hospital stay. Overall, flap morbidity, delayed wound healing, fat necrosis, and flap loss were similar to controls. Conclusions: Arterial and venous anatomies play unique roles in flap reliability. DIEP flap venous congestion must be treated expeditiously with venous augmentation to relieve venous congestion and mitigate flap morbidity.

Internal Mammary Lymph Node Biopsy During Free-Flap Breast Reconstruction: Optimizing Adjuvant Breast Cancer Treatment Through Comprehensive Staging

BackgroundAccurate breast cancer staging is essential for optimal management of adjuvant therapies. While breast lymphatic drainage involves both axillary and internal mammary (IM) lymph node (LN) basins, IM LN sampling is not routinely advocated. The current study analyzes the incidence of IM LN metastases sampled during free flap breast reconstruction and subsequent changes in adjuvant treatment.MethodsA retrospective analysis of patients with positive IM LN biopsies during free flap breast reconstruction was performed. Pre-reconstruction surgical and adjuvant therapies as well as staging and prognostic data were recorded. Change in adjuvant therapies based solely on IM LN positivity was determined.ResultsIM LN metastases were found on 28 (1.3%) out of 2057 patients and comprised the study population. Mean age was 49 years with pre-reconstruction chemotherapy or radiation administered in 50 or 54% of cases, respectively. Five (18%) patients had previously undergone lumpectomy with axillary sampling. Mean tumor size was 3.1 cm with tumor location evenly distributed among all four quadrants. Ten (36%) patients had isolated IM LN metastases Patients with both axillary and IM disease had larger lesions, increased prevalence of pre-reconstruction chemotherapy and radiation. Based exclusively on positive IM LN disease, 17 (63%) patients had a change in adjuvant therapy.ConclusionDespite the low incidence of IM LN metastases, IM LN biopsy during free flap breast reconstruction is recommended. In 36% of cases, nodal metastases were isolated to the IM nodes. Identification of IM metastases influenced adjuvant therapies in a majority of cases.

Positive Margin Re-Excision Following Immediate Autologous Breast Reconstruction: Morbidity, Cosmetic Outcome, and Oncologic Significance

Background Acquisition of negative resection margins is paramount in the surgical management of operable breast cancer. Management of positive margins following mastectomy and immediate breast reconstruction is presently poorly defined. Objectives The present study aims at defining morbidity and cosmetic sequela of re-excision procedures aimed at clearing involved mastectomy margins in the setting of immediate autologous breast reconstruction. Oncologic outcomes are recorded. Methods A retrospective study of patients that underwent skin-sparing mastectomy followed by immediate deep inferior epigastric perforator flap breast reconstruction was performed. Patients found to have positive mastectomy margins underwent margin re-excision during a separate procedure. Method of positive margin exposure and resection is described. Flap morbidity and cosmetic outcome following margin re-excision was compared between reconstructed breasts that underwent re-excision vs those reconstructed after prophylactic mastectomy (controls). Cancer recurrence was recorded during the follow-up period. Results Thirty-six (2.5%) out of 1443 patients were found to have positive mastectomy margins following immediate breast reconstruction between May 2007 and November 2012. Location of positive margins was evenly distributed in all breast regions. Although flap morbidity was similar, a trend (P > 0.05) toward higher seroma formation and fat necrosis was reported in breasts following re-excision vs controls. With a mean follow-up period of 28 months, cosmetic outcome between breasts that underwent re-excision vs controls were similar. Cancer recurrence was reported in 3 (8.3%) patients. Conclusions Re-excision of positive mastectomy margins following immediate autologous breast reconstruction requires a multidisciplinary approach and may be performed with minimal additional morbidity while preserving optimal cosmetic outcome. Level of Evidence 3.

Author Correction: Attenuation of RNA polymerase II pausing mitigates BRCA1-associated R-loop accumulation and tumorigenesis

This corrects the article DOI: 10.1038/ncomms15908.