Krista L. Hardy

Propranolol Accelerates Adipogenesis in Hemangioma Stem Cells and Causes Apoptosis of Hemangioma Endothelial Cells

Background: Infantile hemangiomas can cause significant morbidity during proliferation, yet there is no U.S. Food and Drug Administration–approved treatment. They are believed to form from hemangioma stem cells, which differentiate toward a hemangioma endothelial cell phenotype. Recently, propranolol has demonstrated effectiveness in treating complicated infantile hemangiomas. The authors hypothesize that propranolol facilitates their involution by altering cellular behavior in both hemangioma endothelial and stem cells. Methods: Hemangioma endothelial and stem cells were isolated from resected infantile hemangioma specimens. Cells were treated with 100 &mgr;M propranolol for 48 hours, and apoptosis was determined by the presence of annexin V antibody. Proliferation of stem and endothelial cells was assessed after treatment with 50 or 100 &mgr;M propranolol or vehicle, for 72 and 96 hours, respectively. Adipogenesis was induced in stem cells with and without propranolol. Pro-adipogenic genes PPAR&dgr;, PPAR&ggr;, C/EBP&agr;, C/EBP&bgr;, C/EBP&dgr;, RXR&agr;, and RXR&ggr; were analyzed by quantitative polymerase chain reaction. Results: Annexin V levels were increased in propranolol-treated endothelial cells but not in stem cells. Proliferation of stem and endothelial cells was inhibited by propranolol in a dose-dependent manner. Propranolol-treated stem cells demonstrated accelerated adipogenesis when compared with untreated controls. Transcript levels of C/EBP&bgr; (p < 0.05), RXR&ggr; (p < 0.05), and PPAR&ggr; (p < 0.02) were significantly increased when treated with 50 or 100 &mgr;M propranolol; and C/EBP&dgr; (p < 0.05), RXR&agr; (p < 0.05), and PPAR&dgr; (p < 0.01) transcripts were increased when treated with 100 &mgr;M propranolol. C/EBP&agr; transcript levels remained unchanged at either dose. Conclusions: Propranolol increased apoptosis of hemangioma endothelial cells, but not stem cells, and accelerated adipogenesis of hemangioma stem cells. Thus, propranolol likely accelerates involution to fibrofatty residuum.

Treatment of hyperhidrosis with botulinum toxin.

Botulinum toxin type A is a safe and effective method for treating focal hyperhidrosis, providing longer-lasting results than topical treatments without the necessity of invasive surgical procedures. Although more useful for axillary hyperhidrosis, botulinum toxin injections can also be effective in treating palmar and plantar disease. The effects of botulinum toxin last for six to nine months on average, and treatment is associated with a high satisfaction rate among patients. In this article, the authors discuss their preferred methods for treating axillary, palmar, and plantar hyperhidrosis. This article serves as guide for pretreatment evaluation, injection techniques, and posttreatment care.

Propranolol promotes accelerated and dysregulated adipogenesis in hemangioma stem cells.

BackgroundInfantile hemangiomas (IHs) are the most common tumor of infancy, yet there are no Food and Drug Administration–approved therapeutics to date. Recently, the nonselective &bgr;-adrenergic-blocker propranolol has been shown to be a safe and effective means of treating IHs, although its mechanism has yet to be elucidated. We have previously demonstrated that propranolol induces early and incomplete adipogenesis in stem cells derived from hemangiomas. We hypothesize that propranolol promotes dysregulated adipogenesis via the improper regulation of adipogenic genes. MethodsHemangioma stem cells (HemSCs) isolated from resected IH specimens were treated with adipogenic medium for 1 or 4 days in either propranolol or vehicle. Cell death was measured by the incorporation of annexin V and propidium iodide by flow cytometry. Adipogenesis was assessed by visualizing lipid droplet formation by Oil Red O staining. Proadipogenic genes C/EBP&agr;, C/EBP&bgr;, C/EBP&dgr;, PPAR&dgr;, PPAR&ggr;, RXR&agr;, and RXR&ggr; were analyzed by quantitative reverse transcription and polymerase chain reaction. ResultsHemangioma stem cells treated with propranolol increased lipid droplet formation compared to vehicle-treated cells indicating increased adipogenesis. Cell death as measured by FACS analysis indicated that the propranolol-treated cells died due to necrosis and not apoptosis. During adipogenesis, transcript levels of PPAR&dgr;, PPAR&ggr;, C/EBP&bgr;, and C/EBP&dgr; were significantly increased (P < 0.01) in propranolol-treated cells relative to control cells. In contrast, RXR&agr; and RXR&ggr; levels were significantly decreased (P < 0.05), and C/EBP&agr;, a gene required for terminal adipocyte differentiation, was strongly suppressed by propranolol when compared to vehicle-treated cells (P < 0.01). ConclusionsIn HemSCs, propranolol accelerated dysregulated adipogenic differentiation characterized by improper adipogenic gene expression. Consistent with accelerated adipogenesis, propranolol significantly increased the expression of the proadipogenic genes, PPAR&ggr;, C/EBP&bgr;, and C/EBP&ggr; compared to control. However, propranolol treatment also led to improper induction of PPAR&dgr; and suppression of C/EBP&agr;, RXR&agr;, and RXR&ggr;. Taken together these data indicate that propranolol promoted dysregulated adipogenesis and inhibited the HemSCs from becoming functional adipocytes, ultimately resulting in cell death. Understanding this mechanism behind propranolol’s effectiveness will be a vital factor in producing more effective therapies in the future.

Pulsed Electromagnetic Fields Reduce Postoperative Interleukin-1β, Pain, and Inflammation: A Double-Blind, Placebo-Controlled Study in TRAM Flap Breast Reconstruction Patients.

Background: Pulsed electromagnetic fields have been shown to reduce postoperative pain, inflammation, and narcotic requirements after breast reduction and augmentation surgical procedures. This study examined whether pulsed electromagnetic field therapy could produce similar results in patients undergoing unilateral transverse rectus abdominis myocutaneous (TRAM) flap breast reconstruction, a significantly more complex and painful surgical procedure. Methods: In this double-blind, placebo-controlled, randomized study, 32 patients undergoing unilateral TRAM flap breast reconstruction received active or sham pulsed electromagnetic field therapy. Pain levels were measured by using a visual analogue scale; narcotic use and wound exudate volume were recorded starting 1 hour postoperatively. Wound exudates were analyzed for interleukin-1&bgr;. Results: Mean visual analogue scale pain scores were 2-fold higher in the sham cohort at 5 hours and 4-fold higher at 72 hours (p < 0.01), along with a concomitant 2-fold increase in narcotic use in sham patients (p < 0.01). Wound exudate volume was 2-fold higher in the sham cohort at 24 hours (p < 0.01), and mean interleukin-1&bgr; concentration in wound exudates of sham patients was 5-fold higher at 24 hours (p < 0.001). Conclusions: Pulsed electromagnetic field therapy significantly reduced postoperative pain, inflammation, and narcotic use following TRAM flap breast reconstruction, paralleling its effect in breast reduction patients. Both studies also report a significant reduction of interleukin-1&bgr; in the wound exudate, supporting a mechanism involving a pulsed electromagnetic field effect on nitric oxide/cyclic guanosine monophosphate signaling, which modulates the body’s antiinflammatory pathways. Adjunctive pulsed electromagnetic field therapy could impact the speed and quality of wound repair in many surgical procedures. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, I.

Pulsed electromagnetic fields dosing impacts postoperative pain in breast reduction patients

BACKGROUNDnPulsed electromagnetic fields (PEMF) reduce postoperative pain and narcotic requirements in breast augmentation, reduction, and reconstruction patients. PEMF enhances both calmodulin-dependent nitric oxide and/or cyclic guanosine monophosphate signaling and phosphodiesterase activity, which blocks cyclic guanosine monophosphate. The clinical effect of these competing responses on PEMF dosing is not known.nnnMETHODSnTwo prospective, nonrandomized, active cohorts of breast reduction patients, with 15xa0min PEMF per 2xa0h; Q2 (active), and 5xa0min PEMF per 20xa0min; 5/20 (active), dosing regimens were added to a previously reported double-blind clinical study wherein 20xa0min PEMF per 4xa0h, Q4 (active), dosing significantly accelerated postoperative pain reduction compared with Q4 shams. Postoperative visual analog scale pain scores and narcotic use were compared with results from the previous study.nnnRESULTSnVisual analog scale scores at 24xa0h were 43% and 35% of pain at 1xa0h in the Q4 (active) and Q2 (active) cohorts, respectively (Pxa0<xa00.01). Pain at 24xa0h in the 5/20 (active) cohort was 87% of pain at 1xa0h, compared with 74% in the Q4 (sham) cohort (Pxa0=xa00.451). Concomitantly, narcotic usage in the 5/20 (active) and Q4 (sham) cohorts was not different (Pxa0=xa00.478), and 2-fold higher than the Q4 (active) and Q2 (active) cohorts (Pxa0<xa00.02).nnnCONCLUSIONSnThis prospective study shows Q4/Q2, but not 5/20 PEMF dosing, accelerated postoperative pain reduction compared with historical shams. The 5/20 (active) regimen increases NO 4-fold faster than the Q4 (active) regimen, possibly accelerating phosphodiesterase inhibition of cyclic guanosine monophosphate sufficiently to block the PEMF effect. This study helps define the dosing limits of clinically useful PEMF signals.

A retrospective study to classify surgical indications for infantile hemangiomas

Infantile hemangiomas (IHs) spontaneously involute, but some leave contour deformities necessitating surgical correction. There is a paucity of data reviewing predictive risk factors associated with a need for surgery to guide clinicians when counseling parents. Patients undergoing IH resection by a single surgeon from August 2004 to August 2011 were reviewed to determine patient (age, gender, birth history) and IH characteristics (size, location) associated with surgical intervention. Data were compared to published data from the Hemangioma Investigator Group (HIG). Statistical analysis was performed using Students t-test, odds ratio, and logistic regression analysis. Out of 196 referred patients, 112 underwent surgery. There was a female preponderance (3.5:1). Two-thirds of patients (64.9%) first presented to the surgeon at ≤2 years of age, but most underwent surgery between 2 and 3 years (52.7%; average lag time, 11 months). 18 patients underwent surgery at ≤1 year of age. IH patients with preterm birth history had increased risk for needing surgical intervention (odds ratio 2.124, CI 1.31-3.44; pxa0<xa00.0012). A majority (84.7%) of resected IHs were located on the head or neck, significantly higher than the distribution from the HIG data (62.2%; pxa0<xa00.0001). Resected head and neck IHs were smaller than those below the neck (average, 8.85xa0cm(2) vs. 22.35xa0cm(2), pxa0=xa00.017). Preterm birth is associated with higher risk for requiring surgical intervention. IHs on the head and neck are more likely to be removed when compared to those below the neck, and at a smaller size threshold.

Examining reduction mammaplasty in Hispanic and African American populations: A changing landscape in American plastic surgery

As 21st century physicians, we are witnessing a change in the American landscape with an increased proportion of non-Caucasian patients, particularly Black and Hispanic individuals. The Census Bureau predicts that by 2050, the American population will be one quarter Hispanic, yet the plastic surgical literature rarely addresses this growing population. We aimed to determine if specific factors in this population influence preoperative planning and counseling, guide intra-operative decisions, or affect surgical outcomes. Following approval by the Institutional Review Board at Columbia University Medical Center, the authors performed a retrospective chart review of consecutive breast reduction cases completed at a single center between 2003 and 2007. Patient charts were evaluated for demographic background, pre-operative complaints and counseling, operative techniques, pathology specimens, and postoperative complications and outcomes. The method of breast reduction chosen was determined by projected nipple movement, estimated reduction weight, and patient/surgeon preference (JA, JW, CR). Post-operatively, patients had scheduled office appointments at 1-2 weeks, 6 wks, at 6 months, and then yearly. 161 patients underwent reduction mammaplasty. 116 (72%) of the patients classified themselves as nonCaucasian. (Table 1) All patients were followed for at least one year. The mean age was 35 years for nonCaucasians and 38 years for Caucasians (p Z ns). NonCaucasian women had a significantly higher body-massindex (BMI Z kg/m) and body-surface-area (BSAZ[Height (cm) Weight (kg)]/3600) than their Caucasian counterparts. Non-Caucasian women were more likely to have a history of smoking and have larger brassiere band sizes. Non-Caucasian patients were more likely to complain of

NOTCH3 regulates stem-to-mural cell differentiation in infantile hemangioma

Infantile hemangioma (IH) is a vascular tumor that begins with rapid vascular proliferation shortly after birth, followed by vascular involution in early childhood. We have found that NOTCH3, a critical regulator of mural cell differentiation and maturation, is expressed in hemangioma stem cells (HemSCs), suggesting that NOTCH3 may function in HemSC-to-mural cell differentiation and pathological vessel stabilization. Here, we demonstrate that NOTCH3 is expressed in NG2+PDGFRβ+ perivascular HemSCs and CD31+GLUT1+ hemangioma endothelial cells (HemECs) in proliferating IHs and becomes mostly restricted to the αSMA+NG2loPDGFRβlo mural cells in involuting IHs. NOTCH3 knockdown in HemSCs inhibited in vitro mural cell differentiation and perturbed αSMA expression. In a mouse model of IH, NOTCH3 knockdown or systemic expression of the NOTCH3 inhibitor, NOTCH3 Decoy, significantly decreased IH blood flow, vessel caliber, and αSMA+ perivascular cell coverage. Thus, NOTCH3 is necessary for HemSC-to-mural cell differentiation, and adequate perivascular cell coverage of IH vessels is required for IH vessel stability.

Abstract 18: NOTCH3 KNOCKDOWN IN HEMANGIOMA STEM CELLS ALTERS HEMANGIOMA FORMATION IN A MURINE MODEL

Background: Infantile hemangiomas (IHs) are thought to originate from hemangioma stem cells (HemSC), though IH pathogenesis is poorly understood. Our recent work showed that NOTCH3 is highly expressed in HemSCs and may play a role in IH progression. In cultured HemSCs, NOTCH3 knockdown perturbed adipogenesis and enhanced endothelial cell differentiation. Using a murine model of IH, we compared the development of IHs from control HemSCs (HemSC-scr) to HemSCs with Notch3 shRNA (HemSC-shN3).

Abstract 80: EXAMINING THE ROLE OF MACROPHAGES IN PROLIFERATING INFANTILE HEMANGIOMAS

Background: Infantile hemangiomas (IH) are a common benign endothelial cell tumor thought to be of stem cell origin (HemSC). Histological analysis indicates that IH consist of endothelial cells (HemEC) and associated macrophages. A role for macrophages in IH progression has been postulated, though their function has yet to be characterized. Our study investigates macrophages in hemangioma development using a murine IH model. This model can eventually be used to study the origin and function of IH-associated macrophages.