Ravi F. Sood

Use of metabolomics to trend recovery and therapy after injury in critically ill trauma patients

IMPORTANCE Metabolomics is the broad and parallel study of metabolites within an organism and provides a contemporaneous snapshot of physiologic state. Use of metabolomics in the clinical setting may help achieve precision medicine for those who have experienced trauma, where diagnosis and treatment are tailored to the individual patient. OBJECTIVE To examine whether metabolomics can (1) distinguish healthy volunteers from trauma patients and (2) quantify changes in catabolic metabolites over time after injury. DESIGN, SETTING, AND PARTICIPANTS Prospective cohort study with enrollment from September 2014 to May 2015 at an urban, level 1 trauma center. Included in the study were 10 patients with severe blunt trauma admitted within 12 hours of injury with systolic blood pressure less than 90 mm Hg or base deficit greater than 6 mEq/L and 5 healthy volunteers. Plasma samples (n = 35) were obtained on days 1, 3, and 7, and they were analyzed using mass spectrometry. MAIN OUTCOMES AND MEASURES Principal component analyses, multiple linear regression, and paired t tests were used to select biomarkers of interest. A broad-based metabolite profile comparison between trauma patients and healthy volunteers was performed. Specific biomarkers of interest were oxidative catabolites. RESULTS Trauma patients had a median age of 45 years and a median injury severity score of 43 (interquartile range, 34-50). Healthy fasting volunteers had a median age of 33 years. Compared with healthy volunteers, trauma patients showed oxidative stress on day 1: niacinamide concentrations were a mean (interquartile range) of 0.95 (0.30-1.45) relative units for trauma patients vs 1.06 (0.96-1.09) relative units for healthy volunteers (P = .02), biotin concentrations, 0.43 (0.27-0.58) relative units for trauma patients vs 1.21 (0.93-1.56) relative units for healthy volunteers (P = .049); and choline concentrations, 0.17 (0.09-0.22) relative units for trauma patients vs 0.21 (0.18-0.22) relative units for healthy volunteers (P = .004). Trauma patients showed lower nucleotide synthesis on day 1: adenylosuccinate concentrations were 0.08 (0.04-0.12) relative units for trauma patients vs 0.15 (0.14-0.17) relative units for healthy volunteers (P = .02) and cytidine concentrations were 1.44 (0.95-1.73) relative units for trauma patients vs 1.74 (1.62-1.98) relative units for healthy volunteers (P = .05). From trauma day 1 to day 7, trauma patients showed increasing muscle catabolism: serine levels increased from 42.03 (31.20-54.95) µM to 79.37 (50.29-106.37) µM (P = .002), leucine levels increased from 69.21 (48.36-99.89) µM to 114.16 (92.89-143.52) µM (P = .004), isoleucine levels increased from 20.43 (10.92-27.41) µM to 48.72 (36.28-64.84) µM (P < .001), and valine levels increased from 122.56 (95.63-140.61) µM to 190.52 (136.68-226.07) µM (P = .004). There was an incomplete reversal of oxidative stress. CONCLUSIONS AND RELEVANCE Metabolomics can function as a serial, comprehensive, and potentially personalized tool to characterize metabolism after injury. A targeted metabolomics approach was associated with ongoing oxidative stress, impaired nucleotide synthesis, and initial suppression of protein metabolism followed by increased nitrogen turnover. This technique may provide new therapeutic and nutrition targets in critically injured patients.

Early leukocyte gene expression associated with age, burn size, and inhalation injury in severely burned adults

BACKGROUND In the patient with burn injury, older age, larger percentage of total body surface area (TBS) burned, and inhalation injury are established risk factors for death, which typically results from multisystem organ failure and sepsis, implicating burn-induced immune dysregulation as a contributory mechanism. We sought to identify early transcriptomic changes in circulating leukocytes underlying increased mortality associated with these three risk factors. METHODS We performed a retrospective analysis of the Glue Grant database. From 2003 to 2010, 324 adults with 20% or greater TBS burned were prospectively enrolled at five US burn centers, and 112 provided blood samples within 1 week after burn. RNA was extracted from pooled leukocytes for hybridization onto Affymetrix HU133 Plus 2.0 GeneChips. A multivariate regression model was constructed to determine risk factors for mortality. Testing for differential gene association associated with age, burn size, and inhalation injury was based on linear models using a fold change threshold of 1.5 and false discovery rate of 0.05. RESULTS After adjusting for potential confounders, age greater than 60 years (relative risk [RR], 4.53; 95% confidence interval [CI], 2.93–6.99), burn size greater than 40% TBS (RR, 4.24; 95% CI, 2.61–6.91), and inhalation injury (RR, 2.08; 95% CI, 1.35–3.21) were independently associated with mortality. No genes were differentially expressed in association with age greater than 60 years or inhalation injury. Fifty-one probe sets representing 39 unique genes were differentially expressed in leukocytes from patients with burn size greater than 40% TBS; these genes were associated with platelet activation and degranulation/exocytosis, and gene-set enrichment analysis suggested increased cellular proliferation and down-regulation of proinflammatory cytokines. CONCLUSION Among adults with large burns, older age, increasing burn size, and inhalation injury have a modest effect on the leukocyte transcriptome in the context of the “genomic storm” induced by a 20% or greater than TBS burned. The 39-gene signature we identified may provide novel targets for the development of therapies to reduce morbidity and mortality associated with burns greater than 40% TBS. LEVEL OF EVIDENCE Epidemiologic study, level III.

Targeted metabolic profiling of wounds in diabetic and nondiabetic mice.

While cellular metabolism is known to regulate a number of key biological processes such as cell growth and proliferation, its role in wound healing is unknown. We hypothesized that cutaneous injury would induce significant metabolic changes and that the impaired wound healing seen in diabetes would be associated with a dysfunctional metabolic response to injury. We used a targeted metabolomics approach to characterize the metabolic profile of uninjured skin and full‐thickness wounds at day 7 postinjury in nondiabetic (db/‐) and diabetic (db/db) mice. By liquid chromatography mass spectrometry, we identified 129 metabolites among all tissue samples. Principal component analysis demonstrated that uninjured skin and wounds have distinct metabolic profiles and that diabetes alters the metabolic profile of both uninjured skin and wounds. Examining individual metabolites, we identified 62 with a significantly altered response to injury in the diabetic mice, with many of these, including glycine, kynurenate, and OH‐phenylpyruvate, implicated in wound healing for the first time. Thus, we report the first comprehensive analysis of wound metabolic profiles, and our results highlight the potential for metabolomics to identify novel biomarkers and therapeutic targets for improved wound healing outcomes.

Dermal Fibroblasts from the Red Duroc Pig Have an Inherently Fibrogenic Phenotype: An In Vitro Model of Fibroproliferative Scarring.

Background: The pathophysiology of hypertrophic scarring is unknown in part because of the lack of a robust animal model. Although the red Duroc pig has emerged as a promising in vivo model, the cellular mechanisms underlying Duroc scarring are unknown, and the size and cost of Duroc pigs are obstacles to their use. Given the central role of the dermal fibroblast in scarring, the authors hypothesized that dermal fibroblasts from the Duroc pig exhibit intrinsic differences in key aspects of the fibroblast response to injury compared with those from the Yorkshire pig, a same-species control that heals normally. Methods: Duroc and Yorkshire dermal fibroblasts were isolated from uninjured dorsal skin. Actin stress fibers and focal adhesions were visualized by immunocytochemistry and transmission electron microscopy. Cell migration was measured using a scratch wound-closure assay. Contractile function was assessed by collagen gel contraction. Expression of scarring-related genes was determined by quantitative real-time reverse-transcriptase polymerase chain reaction, and transforming growth factor (TGF)-&bgr;1 protein expression was determined by Western blotting. Results: Duroc dermal fibroblasts display increased adhesion-complex formation, impaired migration, enhanced collagen contraction, and profibrotic gene and protein expression profiles compared with Yorkshire fibroblasts at baseline. In addition, Duroc fibroblasts overexpressed TGF-&bgr;1 and were less responsive to exogenous TGF-&bgr;1. Conclusions: Duroc dermal fibroblasts have inherent myofibroblastic differentiation that may account for the pathologic scarring in these animals. The authors’ data further validate the Duroc model and support Duroc fibroblast cell culture as a simple, inexpensive, reproducible, and biologically tractable in vitro model for the study of fibroproliferative scarring.

Genome-wide Association Study of Postburn Scarring Identifies a Novel Protective Variant.

OBJECTIVE To identify genetic variants associated with the severity of postburn hypertrophic scarring (HTS) using a genome-wide approach. BACKGROUND Risk of severe postburn HTS is known to depend on race, but the genetic determinants of HTS are unknown. METHODS We conducted a genome-wide association study (GWAS) in a prospective cohort of adults admitted with deep-partial-thickness burns from 2007 through 2014. Scar severity was assessed over time using the Vancouver Scar Scale (VSS), and DNA was genotyped with a >500,000-marker array. We performed association testing of single-nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) >0.01 using linear regression of VSS height score on genotype adjusted for patient and injury characteristics as well as population genetic structure. Array-wide significance was based on Bonferroni correction for multiple testing. RESULTS Of 538 patients (median age 40 years, median burn size 6.0% of body surface area), 71% were men and 76% were White. The mean VSS height score was 1.2 (range: 0-3). Of 289,639 SNPs tested, a variant in the CUB and Sushi multiple domains 1 (CSMD1) gene (rs11136645; MAF = 0.49), was significantly associated with decreased scar height (regression coefficient = -0.23, P = 7.9 × 10). CONCLUSIONS In the first published GWAS of HTS, we report that a common intronic variant in the CSMD1 gene is associated with reduced severity of postburn HTS. If this association is confirmed in an independent cohort, investigating the potential role of CSMD1 in wound healing may elucidate HTS pathophysiology.

Melanocortin-1 Receptor Polymorphisms and the Risk of Complicated Sepsis After Trauma: A Candidate Gene Association Study.

Objective: The aim of the study was to determine if melanocortin-1 receptor (MC1R) single nucleotide polymorphisms (SNPs) are associated with complicated sepsis after trauma. Background: Nosocomial infections are an important cause of morbidity and mortality after trauma. Several SNPs in inflammation-related genes have been associated with sepsis. MC1R is an anti-inflammatory mediator that may be involved in the immune response after trauma. Patients and Methods: We genotyped eight common MC1R SNPs in genomic DNA from subjects enrolled in a previously reported prospective cohort study. Subjects were adult trauma patients admitted to the intensive care unit at a Level 1 trauma center (2003–2005). Results: A total of 1,246 subjects were included in the analysis. The majority were male (70%), severely injured (81%), and injured by a blunt mechanism (89%). Forty percent developed sepsis, and 23% developed complicated sepsis, which was defined as sepsis with organ dysfunction. In logistic regression analysis, with adjustments for age, sex, body mass index, injury severity score, red blood cell transfusion requirement, and mechanism of injury, the MC1RR163Q variant (rs885479) was associated with a lower risk of developing complicated sepsis (adjusted odds ratio [ORadj] = 0.48, 95% confidence interval [CI]: 0.28–0.81, P = 0.006). In a subgroup of 511 subjects with genome-wide SNP data, the association between the MC1RR163Q variant and complicated sepsis remained significant after adjusting for genetic substructure (by principal components) and the above clinical factors (ORadj = 0.30, 95% CI: 0.13–0.70, P = 0.005). Conclusions: MC1RR163Q is associated with a lower risk of complicated sepsis after trauma. Therapeutic targeting of MC1R may be beneficial for trauma patients at risk for complicated sepsis.

Comparison of Outcomes in Immediate Implant-Based Breast Reconstruction Versus Mastectomy Alone

Objectives: Immediate implant-based techniques are common practice in post-mastectomy breast reconstruction. Previous studies have shown an increased complication rate in the setting of immediate versus delayed, MD reconstruction. We aimed to quantify any additional risk in complications when implant-based immediate breast reconstruction (IBR) is performed versus mastectomy alone. Materials and Methods: We retrospectively reviewed all IBR cases and all mastectomies without reconstruction from 2007 to 2011. Patient characteristics, operative details, and complication rates were reviewed and analyzed. Results: IBR was performed in 315 consecutive women; mastectomy alone was performed in 401 women. Patients undergoing mastectomy alone were more often older, diabetic, and more frequently underwent neoadjuvant chemotherapy or radiation. Overall complications were higher in the IBR group, most commonly reoperation and delayed wound healing. In a multivariate analysis, IBR, increasing age, body mass index, history of radiation therapy, smoking, and nipple-sparing mastectomy were independently associated with increased risk of complications. However, IBR was only independently associated with increased risk of major complications such as reoperation or readmission for intravenous antibiotics, not minor complications. Conclusion: Patients selected for IBR are inherently different than those undergoing mastectomy alone. After adjusting for these differences, the increased risk of complications seen in IBR is moderately increased over the risk of complications in mastectomy alone. The observed increased risk of major complications after IBR is largely due to the aggressive management of complications in the setting of a prosthetic implant. IBR is a safe reconstructive strategy with only a slightly increased risk over mastectomy alone.

Race Does Not Predict Melanocyte Heterogeneous Responses to Dermal Fibroblast-Derived Mediators.

Introduction Abnormal pigmentation following cutaneous injury causes significant patient distress and represents a barrier to recovery. Wound depth and patient characteristics influence scar pigmentation. However, we know little about the pathophysiology leading to hyperpigmentation in healed shallow wounds and hypopigmentation in deep dermal wound scars. We sought to determine whether dermal fibroblast signaling influences melanocyte responses. Methods and Materials Epidermal melanocytes from three Caucasians and three African-Americans were genotyped for single nucleotide polymorphisms (SNPs) across the entire genome. Melanocyte genetic profiles were determined using principal component analysis. We assessed melanocyte phenotype and gene expression in response to dermal fibroblast-conditioned medium and determined potential mesenchymal mediators by proteome profiling the fibroblast-conditioned medium. Results Six melanocyte samples demonstrated significant variability in phenotype and gene expression at baseline and in response to fibroblast-conditioned medium. Genetic profiling for SNPs in receptors for 13 identified soluble fibroblast-secreted mediators demonstrated considerable heterogeneity, potentially explaining the variable melanocyte responses to fibroblast-conditioned medium. Discussion Our data suggest that melanocytes respond to dermal fibroblast-derived mediators independent of keratinocytes and raise the possibility that mesenchymal-epidermal interactions influence skin pigmentation during cutaneous scarring.