June K. Wu

Notch1 Deficiency Results in Decreased Inflammation during Wound Healing and Regulates Vascular Endothelial Growth Factor Receptor-1 and Inflammatory Cytokine Expression in Macrophages

We investigated whether Notch signaling plays a role in regulating macrophage responses to inflammation. In a wound healing assay, macrophage recruitment was decreased in Notch1+/− mice, and the wounds were characterized by decreased TNF-α expression. As wound healing progressed, Notch1+/− wounds had increased vascularization and collagen deposition compared with wild-type wounds. In mice with myeloid-specific Notch1 deletion, wounds had decreased macrophage recruitment as well as decreased TNF-α expression, indicating the specific role of Notch1 in the inflammatory response in these cells. In vitro, we found that vascular endothelial growth factor receptor-1 (VEGFR-1) was upregulated in macrophages in response to LPS/IFN-γ and that this upregulation depended on Notch signaling. Furthermore, macrophages from Notch1+/− mice had decreased expression of VEGFR-1 compared with macrophages from wild-type mice, whereas VEGFR-1 expression in Notch4−/− macrophages was normal. Inhibition of Notch signaling decreased induction of the inflammatory cytokines IL-6, IL-12, CXCL10, MCP-1, monokine induced by IFN-γ, and TNF-α in macrophages in response to LPS/IFN-γ. Additionally, macrophages from Notch1+/− mice demonstrated decreased induction of IL-6, IL-12, and TNF-α in response to stimulation compared with wild-type mice. Thus, both pharmacological inhibition of Notch and genetic analysis demonstrate that Notch1 regulates VEGFR-1 and cytokine expression in macrophages. We have also established that Notch1 is important for the inflammatory response during wound healing in mice.

Repair of bilateral cleft lip: review, revisions, and reflections.

Rarely does the appearance of a child with a repaired bilateral cleft lip compare favorably with that of a child with a repaired unilateral cleft lip. However, there has been a major change in operative strategy during the past decade, and as a result, the typical bilateral cleft nasolabial stigmata are no longer so obvious. The senior author restates the principles for correction of bilateral cleft lip and nasal deformity, and underscores the essential role of preoperative premaxillary positioning. He reviews his method of single-stage closure of the cleft primary palate, including three-dimensional adjustments based on predicted four-dimensional changes. Operative modifications are described for variations of bilateral cleft lip. The authors emphasize the surgeons obligation for periodic assessment. In a consecutive series of 50 patients with repaired bilateral complete cleft lip/palate, the revision-rate was 33% as compared with 12.5% if the secondary palate is intact. No revisions were necessary for philtral size or columellar length. The authors propose that nasolabial appearance and speech are the priorities in habilitation of the child with bilateral cleft lip/palate rather than the traditional emphasis on maxillary growth.

Efficacy and mechanisms of vacuum-assisted closure (VAC) therapy in promoting wound healing: a rodent model

BACKGROUND The vacuum-assisted closure device (VAC) has revolutionised wound care, although molecular mechanisms are not well understood. We hypothesise that the VAC device induces production of pro-angiogenic factors and promotes formation of granulation tissue and healing. METHODS A novel rodent model of VAC wound healing was established. Excisional wounds were created on rat dorsa. Wounds were dressed with Tegaderm (control group), VAC Granulofoam and Tegaderm (special control group), or VAC Granulofoam, T.R.A.C. PAD((R)) with 125 mm Hg continuous negative pressure (VAC group). Wound closure rates were calculated as a percentage of initial wound sizes. Rats were sacrificed on postoperative days 3, 5 and 7; harvested tissues were processed for histology [haematoxylin & eosin (H&E), Massons trichrome, picrosirius red] and Western blot analysis (CD31, vascular endothelial growth factor, basic fibroblast growth factor). RESULTS Statistically significant wound closure rates were achieved in the experimental group at all measured time points: day 3, 28.1% (VAC) vs 8.2% (control) and 8.8% (special control) (ANOVA, P<0.0001); day 5, 45.3% (VAC) vs 23.7% (control) and 22.5% (special control) (ANOVA, P=0.0003); day 7, 54.4% (VAC) vs 43.0% (control) and 31.5% (special control) (ANOVA; P<0.0001). Morphological evaluation by Massons trichrome stain showed increased collagen organisation and wound maturation in the VAC group. These wounds also showed increased expression of vascular endothelial growth factor and fibroblast growth factor-2 on day 5 by Western blot analysis. CONCLUSION A small animal VAC wound model was established. Wounds treated with a VAC device showed accelerated wound closure rates, increased pro-angiogenic growth factor production and improved collagen deposition. Further application of this model may elucidate other mechanisms.

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.

JAGGED1 Signaling Regulates Hemangioma Stem Cell–to–Pericyte/Vascular Smooth Muscle Cell Differentiation

Objective—The aim of our study is to determine the cellular and molecular origin for the pericytes in infantile hemangioma (IH) and their functional role in the formation of pathological blood vessels. Methods and Results—Here we show that IH-derived stem cells (HemSCs) form pericyte-like cells. With in vitro studies, we demonstrate that HemSC-to-pericyte differentiation depends on direct contact with endothelial cells. JAGGED1 expressed ectopically in fibroblasts was sufficient to induce HemSCs to acquire a pericyte-like phenotype, indicating a critical role for JAGGED1. In vivo, we blocked pericyte differentiation with recombinant JAGGED1, and we observed reduced formation of blood vessels, with an evident lack of pericytes. Silencing JAGGED1 in the endothelial cells reduced blood vessel formation and resulted in a paucity of pericytes. Conclusion—Our data show that endothelial JAGGED1 controls HemSC-to-pericyte differentiation in a murine model of IH and suggests that pericytes have a fundamental role in formation of blood vessels in IH.

A switch in Notch gene expression parallels stem cell to endothelial transition in infantile hemangioma

BackgroundInfantile hemangioma (IH) is the most common benign tumor of infancy, yet its pathogenesis is poorly understood. Notch family members are known to play a role in vascular development during embryogenesis and postnatal tumor angiogenesis, yet the role of Notch signaling in the pathogenesis of IH has not been investigated. This study aims to survey Notch expression in IH.Materials and methodsRNA from resected hemangioma tissue and hemangioma-derived stem cells (HemSCs) and endothelial cells (HemECs) was used for gene expression analyses by real-time PCR. Results were confirmed with immunofluorescence for protein expression in tissue.ResultsReal-time PCR showed that Notch family gene expression in IH is distinct from placenta and skin. Notch3 is expressed in HemSCs, but not in HemECs, indicating Notch3 is downregulated as HemSCs differentiate into HemECs. Moreover, expression of endothelial-associated Notch proteins, Notch1, Notch4, and Jagged-1 are increased in involuting hemangiomas and HemECs, suggesting that as hemangioma progresses toward involution, it acquires more differentiated endothelium. A subset of cells stained double positive for Notch3 and CD31, pointing to a potential intermediate between the HemSC cellular differentiation into HemEC.ConclusionHemSCs have distinct Notch expression patterns from differentiated HemECs and from normal human endothelial cells. Notch3 is expressed in HemSCs, while Notch1, Notch4, and Jagged-1 have higher expression levels in HemECs. Notch3 was localized to the interstitial cells outside of the nascent vascular channels in proliferating IH tissue sections, but became more apparent in the perivascular cells in involuting IH. In summary, the pattern of Notch gene expression mirrors the progression from immature cells to endothelial-lined vascular channels (i.e., endothelial differentiation) that characterizes the growth and involution of IH.

Craniofacial Growth in Patients With FGFR3Pro250Arg Mutation After Fronto-Orbital Advancement in Infancy

Background: The facial features of children with FGFR3Pro250Arg mutation (Muenke syndrome) differ from those with the other eponymous craniosynostotic disorders. We documented midfacial growth and position of the forehead after fronto-orbital advancement (FOA) in patients with the FGFR3 mutation. Methods: We retrospectively reviewed all patients who had an FGFR3Pro250Arg mutation and craniosynostosis. Only patients who had FOA in infancy or early childhood were included. The clinical records were evaluated for type of sutural fusion; midfacial hypoplasia and other clinical data, including age at operation; type of procedures and fixation (wire vs resorbable plate); frequency of frontal readvancement, forehead augmentation, midfacial advancement; and complications. Preoperative and postoperative sagittal orbital-globe relationship was measured by direct anthropometry. Outcome of FOA was graded according to the Whittaker classification as category I, no revision; category II, minor revisions, that is, foreheadplasty; category III, alternative bony work; category IV; redo of initial procedure (ie, secondary FOA). Midfacial position was determined by clinical examination and lateral cephalometry. Results: A total of 21 study patients with Muenke syndrome (8 males and 13 females) were analyzed. The types of craniosynostosis were bilateral coronal (n = 15), of which 3 also had concurrent sagittal fusion, and unilateral coronal (n = 5). Two patients had early endoscopic suturectomy, but later required FOA. Mean age at FOA was 22.9 months (range, 3-128 months). Secondary FOA was necessary in 40% of patients (n = 8), and secondary foreheadplasty in 25% (n = 5) of patients. No frontal revisions were needed in the remaining 35% of patients (n = 7). Mean age at initial FOA was significantly younger in the group requiring repeat FOA or foreheadplasty compared with patients who did not require revision (P < 0.05). Location of synostosis, type of fixation, and bone grafting did not significantly affect the need for revision. Only 30% (n = 6) of patients developed midfacial retrusion. Conclusions: The frequency of frontal revision in patients with Muenke syndrome who had FOA in infancy and early childhood is lower than previously reported. Age at forehead advancement inversely correlated with the incidence of relapse and need for secondary frontal procedures. Midfacial retrusion is relatively uncommon in FGFR3Pro250Arg patients.

Expression of HES and HEY genes in infantile hemangiomas

BackgroundInfantile hemangiomas (IHs) are the most common benign tumor of infancy, yet their pathogenesis is poorly understood. IHs are believed to originate from a progenitor cell, the hemangioma stem cell (HemSC). Recent studies by our group showed that NOTCH proteins and NOTCH ligands are expressed in hemangiomas, indicating Notch signaling may be active in IHs. We sought to investigate downstream activation of Notch signaling in hemangioma cells by evaluating the expression of the basic HLH family proteins, HES/HEY, in IHs.Materials and MethodsHemSCs and hemangioma endothelial cells (HemECs) are isolated from freshly resected hemangioma specimens. Quantitative RT-PCR was performed to probe for relative gene transcript levels (normalized to beta-actin). Immunofluorescence was performed to evaluate protein expression. Co-localization studies were performed with CD31 (endothelial cells) and NOTCH3 (peri-vascular, non-endothelial cells). HemSCs were treated with the gamma secretase inhibitor (GSI) Compound E, and gene transcript levels were quantified with real-time PCR.ResultsHEY1, HEYL, and HES1 are highly expressed in HemSCs, while HEY2 is highly expressed in HemECs. Protein expression evaluation by immunofluorescence confirms that HEY2 is expressed by HemECs (CD31+ cells), while HEY1, HEYL, and HES1 are more widely expressed and mostly expressed by perivascular cells of hemangiomas. Inhibition of Notch signaling by addition of GSI resulted in down-regulation of HES/HEY genes.ConclusionsHES/HEY genes are expressed in IHs in cell type specific patterns; HEY2 is expressed in HemECs and HEY1, HEYL, HES1 are expressed in HemSCs. This pattern suggests that HEY/HES genes act downstream of Notch receptors that function in distinct cell types of IHs. HES/HEY gene transcripts are decreased with the addition of a gamma-secretase inhibitor, Compound E, demonstrating that Notch signaling is active in infantile hemangioma cells.

Management of lip hemangiomas: Minimizing peri-oral scars☆

PURPOSE Hemangiomas are the most common benign tumor of infancy, affecting females more than males. Lip hemangiomas are of particular concern because of their relatively increased risk to ulcerate during the proliferative period. Ulcerated hemangiomas of the lip can lead to increased scarring, loss of lip contour, and disfigurement. Most will require surgical correction to restore normal labial anatomy. METHODS A retrospective chart review between 2004 and 2010 for surgically treated lip hemangiomas was performed. Demographic data, location of the hemangioma, age at operation, and number of operations were recorded. Two independent observers evaluated lip appearance post-operatively using 5-point scales to examine scar, symmetry, contour, and color, with 5 being excellent and 1 being poor. RESULTS Between 2004 and 2010, eleven patients underwent surgical correction. Ten of the eleven were female. 18% (2/11) were ulcerated. One third (4/11) was in the upper lip and two-thirds (7/11) were in the lower lip. The mean age of the patients at the time of operation was 3.6 years (range, 14 months to 17 years). The average number of operations per patient was 1.6 (range, 1-3). The average scores for lip appearance after surgical correction ranged between 3.95 (good) for lip contour to 4.5 (good to excellent) for color. CONCLUSIONS Lip hemangiomas often require surgical correction. Treatment goals include restoration of normal lip contour and strategic placement of the incision. By taking advantage of the natural involution that occurs and careful planning, procedures can be staged to minimize distortion of the lip. Even lip hemangiomas that cross the vermilio-cutaneous (VC) junction can be excised and lip contour achieved without the need to extend scars beyond the VC junction.

Aberrant Lymphatic Endothelial Progenitors in Lymphatic Malformation Development

Lymphatic malformations (LMs) are vascular anomalies thought to arise from dysregulated lymphangiogenesis. These lesions impose a significant burden of disease on affected individuals. LM pathobiology is poorly understood, hindering the development of effective treatments. In the present studies, immunostaining of LM tissues revealed that endothelial cells lining aberrant lymphatic vessels and cells in the surrounding stroma expressed the stem cell marker, CD133, and the lymphatic endothelial protein, podoplanin. Isolated patient-derived CD133+ LM cells expressed stem cell genes (NANOG, Oct4), circulating endothelial cell precursor proteins (CD90, CD146, c-Kit, VEGFR-2), and lymphatic endothelial proteins (podoplanin, VEGFR-3). Consistent with a progenitor cell identity, CD133+ LM cells were multipotent and could be differentiated into fat, bone, smooth muscle, and lymphatic endothelial cells in vitro. CD133+ cells were compared to CD133− cells isolated from LM fluids. CD133− LM cells had lower expression of stem cell genes, but expressed circulating endothelial precursor proteins and high levels of lymphatic endothelial proteins, VE-cadherin, CD31, podoplanin, VEGFR-3 and Prox1. CD133− LM cells were not multipotent, consistent with a differentiated lymphatic endothelial cell phenotype. In a mouse xenograft model, CD133+ LM cells differentiated into lymphatic endothelial cells that formed irregularly dilated lymphatic channels, phenocopying human LMs. In vivo, CD133+ LM cells acquired expression of differentiated lymphatic endothelial cell proteins, podoplanin, LYVE1, Prox1, and VEGFR-3, comparable to expression found in LM patient tissues. Taken together, these data identify a novel LM progenitor cell population that differentiates to form the abnormal lymphatic structures characteristic of these lesions, recapitulating the human LM phenotype. This LM progenitor cell population may contribute to the clinically refractory behavior of LMs.