Jennifer E. Bond

Pseudomonas aeruginosa PilY1 binds integrin in an RGD- and calcium-dependent manner.

PilY1 is a type IV pilus (tfp)-associated protein from the opportunistic pathogen Pseudomonas aeruginosa that shares functional similarity with related proteins in infectious Neisseria and Kingella species. Previous data have shown that PilY1 acts as a calcium-dependent pilus biogenesis factor necessary for twitching motility with a specific calcium binding site located at amino acids 850–859 in the 1,163 residue protein. In addition to motility, PilY1 is also thought to play an important role in the adhesion of P. aeruginosa tfp to host epithelial cells. Here, we show that PilY1 contains an integrin binding arginine-glycine-aspartic acid (RGD) motif located at residues 619–621 in the PilY1 from the PAK strain of P. aeruginosa; this motif is conserved in the PilY1s from the other P. aeruginosa strains of known sequence. We demonstrate that purified PilY1 binds integrin in vitro in an RGD-dependent manner. Furthermore, we identify a second calcium binding site (amino acids 600–608) located ten residues upstream of the RGD. Eliminating calcium binding from this site using a D608A mutation abolished integrin binding; in contrast, a calcium binding mimic (D608K) preserved integrin binding. Finally, we show that the previously established PilY1 calcium binding site at 851–859 also impacts the proteins association with integrin. Taken together, these data indicate that PilY1 binds to integrin in an RGD- and calcium-dependent manner in vitro. As such, P. aeruginosa may employ these interactions to mediate host epithelial cell binding in vivo.

Wound contraction is attenuated by fasudil inhibition of Rho-associated kinase.

Background: Dermal scarring and scar contracture result in restriction of movement. There are no effective drugs to prevent scarring. RhoA and Rho-associated kinase have emerged as regulators of fibrosis and contracture. Fasudil, a Rho-associated kinase inhibitor, has been demonstrated to have antifibrotic effects in models of liver, renal, and cardiac fibrosis. The role of fasudil in preventing dermal scarring and contractures has not been studied. The authors used a rat model of dermal wound healing to assess the effects of fasudil with regard to the prevention of scarring. Methods: Human scar tissue and surrounding normal skin were immunostained for RhoA and Rho-associated kinase. Full-thickness wounds were created on Wistar-Han rats, and fasudil (30 mg/kg/day) or saline was continuously delivered subcutaneously. Wound contraction was measured by gravitational planimetry. After 21 days, tissue was harvested for Massons trichrome, hematoxylin and eosin, Ki-67, and CD31 staining. Fibroblast-populated collagen lattices were used to assess the mechanistic effects of fasudil on contractility. Myofibroblast formation was assessed in the presence of fasudil. Results: Human scar tissue in the remodeling phase of repair showed increased expression of RhoA and Rho-associated kinase in scar tissue compared with surrounding normal tissue. Fasudil inhibited wound contraction as compared with controls. Hematoxylin and eosin and Massons trichrome were similar between groups. Fasudil did not alter angiogenesis or proliferation. Fasudil inhibited fibroblast contractility and myofibroblast formation in vitro. Conclusions: There is growing evidence that the RhoA/Rho-associated kinase pathway plays an important role in wound healing and scar contracture. The authors present data showing that inhibition of Rho-associated kinase hinders fibroblast contractility and may be beneficial in preventing scar contracture.

Temporal spatial expression and function of non-muscle myosin II isoforms IIA and IIB in scar remodeling

Scar contracture is believed to be caused by the cell contractility during the remodeling phase of wound healing. Cell contractility is mediated by non-muscle myosin II (NMMII) and actin, but the temporal-spatial expression profile of NMMII isoforms A and B (IIA and IIB) during the remodeling phase and the role of NMMII in scar fibroblast tissue remodeling are unknown. Human scar tissue immunostained for IIA and IIB showed that both isoforms were highly expressed in scar tissue throughout the remodeling phase of repair and expression levels returned to normal after the remodeling phase. Human scar tissue immunostained for β-, γ- and α–smooth muscle actin showed that all isoforms were consistently expressed throughout the remodeling phase of repair. The β- and γ-smooth muscle actin were widely expressed throughout the dermis, but α-smooth muscle actin was only locally expressed within the dermis. In vitro, fibroblasts explanted from scar tissue were shown to express more IIA than fibroblasts explanted from normal tissue and scar fibroblasts contracted collagen lattices to a greater extent than normal fibroblasts. Blebbistatin was used to demonstrate the function of NMMII in collagen lattice contraction. In normal tissue, fibroblasts are stress-shielded from external tensile stress by the extracellular matrix. After dermal injury and during remodeling, fibroblasts are exposed to a matrix of increased stiffness. The effect of matrix stiffness on IIA and IIB expression was examined. IIA expression was greater in fibroblasts cultured in collagen lattices with increasing stiffness, and in fibroblasts cultured on glass slides compared with polyacrylamide gels with stiffness of 1 kPa. In conclusion, NMMII and actin isoform expression changes coordinately with the remodeling phase of repair, and NMMII is increased as matrix stiffness increases. As NMMII expression increases, so does the fibroblast contractility.

The Modified Patient and Observer Scar Assessment Scale: A Novel Approach to Defining Pathologic and Nonpathologic Scarring?

Background: Scarring is a highly prevalent and multifactorial process, yet no studies to date have attempted to distinguish pathologic from nonpathologic scarring. Methods: This article defines and proposes methods of classifying pathologic scarring as it pertains to clinical presentation. Results: The authors propose a new scar scale that incorporates pain and functional impairment. Conclusions: The modified Patient and Observer Scar Assessment Scale is the first of its kind to factor in the functional deficits pain and pruritus of scarring into measurements of associated morbidity. This scale has great potential in evaluating patient response to treatment and analyzing clinical outcomes.

A Detailed Evaluation of the Anatomical Variations of the Profunda Artery Perforator Flap Using Computed Tomographic Angiograms

Background: The profunda artery perforator flap is a new option for breast reconstruction in appropriate patients. While the basic anatomy is known, detailed profunda perforator anatomy has never fully been described and we present new data that will aid dissection. Methods: Fifty consecutive lower extremity computed tomography angiogram scans (100 legs) were retrospectively analyzed to acquire profunda artery perforator measurements. Patient medical records were then examined to ascertain patient information. Data were then analyzed using simple descriptive statistics and bivariate linear regressions with repeated measures. Results: Bilateral thighs from 50 consecutive angiograms were included for a total of 100 thighs. Females comprised 30 (60 percent) of the patients and the cohort average age was 59.1 years old. All thighs had at least two perforators, with 85 percent having three or more. On average, perforators were located 6.2 cm below the gluteal crease, and were evenly distributed between the medial and lateral halves of the thigh. The average perforator diameter at origin off profunda was 2.7 mm. There was significantly greater diameter in vessels in the lateral thigh (p < 0.001), in patients with higher Body Mass Index (BMI) (p < 0.05), and in patients with decreased age (p < 0.05). Males were more likely to have perforators that shared a common trunk off the profunda artery (p < 0.05). Conclusions: At least two profunda perforators exist in each thigh with an average diameter suitable for microvascular transfer, although larger perforators are observed laterally and in younger patients with higher BMI.

Antigen delivery by α2-macroglobulin enhances the cytotoxic T lymphocyte response

α2M* targets antigens to APCs for rapid internalization, processing, and presentation. When used as an antigen‐delivery vehicle, α2M* amplifies MHC class II presentation, as demonstrated by increased antibody titers. Recent evidence, however, suggests that α2M* encapsulation may also enhance antigen‐specific CTL immunity. In this study, we demonstrate that α2M*‐delivered antigen (OVA) enhances the production of specific in vitro and in vivo CTL responses. Murine splenocytes expressing a transgenic TCR specific for CTL peptide OVA257–264 (SIINFEKL) demonstrated up to 25‐fold greater IFN‐γ and IL‐2 secretion when treated in vitro with α2M*‐OVA compared with soluble OVA. The frequency of IFN‐γ‐producing cells was increased ∼15‐fold, as measured by ELISPOT. Expansion of the OVA‐specific CD8+ T cell population, as assayed by tetramer binding and [3H]thymidine incorporation, and OVA‐specific cell‐mediated cytotoxicity, as determined by a flow cytometric assay, were also enhanced significantly by α2M*‐OVA. Furthermore, significant CTL responses were observed at antigen doses tenfold lower than those required with OVA alone. Finally, we also observed enhanced humoral and CTL responses by naïve mice following intradermal immunization with α2M*‐OVA. These α2M*‐OVA‐immunized mice demonstrated increased protection against a s.c.‐implanted, OVA‐expressing tumor, as demonstrated by delayed tumor growth and prolonged animal survival. The observation that α2M*‐mediated antigen delivery elicits specific CTL responses suggests the cross‐presentation of antigen onto MHC class I. These results support α2M* as an effective antigen‐delivery system that may be particularly useful for vaccines based on weakly immunogenic subunits or requiring dose sparing.

Myofibroblasts contribute to but are not necessary for wound contraction.

Wound contraction facilitates tissue repair. The correct balance between too little contraction, which leads to non-healing wounds, and too much contraction, which leads to contractures, is important for optimal healing. Thus, understanding which cells cause wound contraction is necessary to optimize repair. Wound contraction is hypothesized to develop from myofibroblast (cells which express alpha-smooth muscle actin; ACTA2) contractility, while the role of fibroblast contractility is unknown. In this study, we utilized ACTA2 null mice to determine what role fibroblasts play in wound contraction. Human scar contractures were immunostained for ACTA2, beta-cytoplasmic actin (ACTB), and gamma-cytoplasmic actin (ACTG1). Full-thickness cutaneous wounds were created on dorsum of ACTA2+/+ mice and strain-matching ACTA2+/− and ACTA2−/− mice. Wound contraction was quantified. Tissue was harvested for histologic, immunohistochemical and protein analysis. Compared with surrounding unwounded skin, human scar tissue showed increased expression of ACTA2, ACTB, and ACTG1. ACTA2 was focally expressed in clusters. ACTB and ACTG1 were widely, highly expressed throughout scar tissue. Wound contraction was significantly retarded in ACTA2−/− mice, as compared to ACTA2+/+ controls. Control mice had increased epithelialization, cell proliferation, and neovascularization. ACTA2−/− mice had lower levels of apoptosis, and fewer total numbers of cells. Smaller amount of collagen deposition and immature collagen organization in ACTA2−/− mice demonstrate that wounds were more immature. These data demonstrate that myofibroblasts contribute to but are not necessary for wound contraction. Mechanisms by which fibroblasts promote wound contraction may include activation of contractile signaling pathways, which promote interaction between non-muscle myosin II and ACTB and ACTG1.

The Role of Osteopontin and Osteopontin Aptamer (OPN-R3) in Fibroblast Activity

BACKGROUND Scarring is believed to be caused by both persistent inflammation and overexuberant fibroblast activation. Osteopontin (OPN) is a cytokine that promotes cell activation. The absence of OPN in vivo reduces dermal scarring. This suggests that OPN is involved in scar formation; however, how OPN exerts these pro-scarring effects is unknown. RNA aptamers are short RNA molecules that bind target proteins with high affinity. The aptamer OPN-R3 (R3) blocks OPN signaling. The role of R3 in preventing dermal fibrosis is unknown. METHODS Fibroblast migration was analyzed with the use of Boyden Chambers and HEMA-3 staining. Inverted confocal microscopy was used to assess fibroblast focal adhesion length. Adhesion was measured by incubating fluorescently stained fibroblasts on OPN coated 96-well plates. CellTiter 96 AQueous non-radioactive cell proliferation assay was utilized to investigate the proliferative activity of fibroblasts. Free floating collagen lattices were utilized to assess fibroblast contractility. RESULTS Human dermal fibroblasts migrated significantly in response to OPN. OPN did not induce a significant increase in focal adhesion length compared with controls. Adhesion studies demonstrated that OPN increased fibroblast adhesion. Proliferation assays indicate that OPN increased fibroblast growth. OPN increased fibroblast contractility of collagen lattices. The addition of R3 significantly inhibited OPN-induced activity. CONCLUSION OPN is associated with scar and exerts pro-scarring effects by increasing cellular migration, adhesion, proliferation, and contractility of human dermal fibroblasts. R3 prevents OPN mediated activity. OPN may be useful for promoting closure of non-healing wounds and the OPN specific aptamer, R3, may be useful for preventing fibrosis.

Changes in dermal histomorphology following surgical weight loss versus diet-induced weight loss in the morbidly obese patient.

Introduction: Patients with postgastric bypass and diet-induced weight loss present to the plastic surgeon for various body contouring procedures. Gross differences in skin dermal elasticity may exist between these populations; however, studies evaluating histologic differences are lacking. This prospective study aims to evaluate histomorphologic differences in morbidly obese patients following surgical versus diet-induced (nonsurgical) weight loss. Further, we aim to elicit if postoperative complications are correlated with the mechanism of weight loss and potential histomorphologic differences. Methods: Defined infraumbilical skin specimens were collected during abdominal contouring procedures following weight loss achieved through surgical or nonsurgical means. Specimens were stained for elastic fiber content and morphology, collagen deposition, and inflammation. All sections underwent evaluation for quality and quantity of elastic fibers, collagen architecture, and presence of inflammation in the context of age-matched controls. Histomorphological results were compared between the 2 groups and subanalyzed according to clinical variables and postbody contouring wound complications. Results: Between July 2008 and December 2010, 30 consecutive patients with significant weight loss (17 surgical, 13 nonsurgical) underwent a panniculectomy (n = 15), abdominoplasty (n = 13), and lower body lift (n = 2), with an average age of 48.3 ± 11.10 years and a body mass index of 39.23 ± 13.65 kg/m2. Demographic and clinical variables were not statistically significant between the 2 groups. Blinded histologic evaluation revealed a trend toward normal elastic fiber appearance (P = 0.255), increased wound complications (P = 0.546), and mild inflammation (P = 0.462) in the surgical group. Analysis of dermal histomorphology correlating with wound complications was not statistically significant at follow-up (4.76 ± 5.55 months). Interestingly, there was a persistent inflammatory component in both groups when compared with age-matched controls. Conclusions: Although the differences in histomorphology between the surgical and nonsurgical weight loss groups did not reach statistical significance, the results demonstrated an existence of weight loss-induced histomorphological skin changes that may impact future studies. The study did not demonstrate a relationship between dermal histomorphology and postoperative wound complications, suggesting that aberrant healing in body contouring procedures involves a multifactorial process.

Dupuytren's fibroblast contractility by sphingosine-1-phosphate is mediated through non-muscle myosin II.

PURPOSE Previous studies suggest that Dupuytrens disease is caused by fibroblast and myofibroblast contractility within Dupuytrens nodules; however, the stimulus for cell contractility is unknown. Sphingosine-1-phosphate (S1P) is a serum-derived lysophospholipid mediator that enhances cell contractility by activating the S1P receptor, S1P(2). It is hypothesized that S1P stimulates Dupuytrens fibroblast contractility through S1P(2) activation of non-muscle myosin II (NMMII). This investigation examined the role of S1P and NMMII activation in Dupuytrens disease progression and suggests potential targets for treatment. METHODS We enmeshed Dupuytrens fibroblasts into fibroblast-populated collagen lattices (FPCLs) and assayed S1P-stimulated FPCL contraction in the presence of the S1P(2) receptor inhibitor JTE-013, the Rho kinase inhibitor Y-27632, the myosin light chain kinase inhibitor ML-7, and the NMMII inhibitor blebbistatin. Tissues from Dupuytrens fascia (n = 10) and normal palmar fascia (n = 10) were immunostained for NMMIIA and NMMIIB. RESULTS Sphingosine-1-phosphate stimulated FPCL contraction in a dose-dependent manner. Inhibition of S1P(2) and NMMII prevented S1P-stimulated FPCL contraction. Rho kinase and myosin light chain kinase inhibited both S1P and control FPCL contraction. Dupuytrens nodule fibroblasts robustly expressed NMMIIA and NMMIIB, compared with quiescent-appearing cords and normal palmar fascia. CONCLUSIONS Sphingosine-1-phosphate promotes Dupuytrens fibroblast contractility through S1P(2), which stimulates activation of NMMII. NMMII isoforms are ubiquitously expressed throughout Dupuytrens nodules, which suggests that nodule fibroblasts are primed to respond to S1P stimulation to cause contracture formation. S1P-promoted activation of NMMII may be a target for disease treatment.