Unilateral Cleft Lip Repair Using the Chang Gung Technique: An Outcome Analysis.

OBJECTIVE: This retrospective study analyzed the outcomes of patients undergoing complete or incomplete unilateral cleft lip repair using the Chang Gung technique. The goal was to compare the symmetry and change of the technique through the measurement of anthropometric points on digital photographs. METHODS: From 2010 to 2016, a total of 274 complete and incomplete cleft lip patients without other craniofacial deformities were included in the study. All included patients had a minimum 1-year follow-up with frontal view photographs taken. The vermilion area, lip width, vermilion height, lateral lip length, lip height, and Cupid's bow width of both cleft and noncleft sides were measured for all patients. The Cleft Lip Component Symmetry Index was used to determine the symmetry of the cleft and noncleft sides in both incomplete and complete cleft groups. RESULTS: A total of 152 complete and 122 incomplete cleft lip patients were included in the study. The mean Cleft Lip Component Symmetry Index values showed that the vermilion area, lip height, and Cupid's bow width were symmetric on both cleft and noncleft sides. Lip width and lateral lip length were noted to be shorter, whereas the vermilion height was thicker on the cleft side than on the noncleft side. Significant differences between the complete and incomplete cleft group measurements were found for lateral lip length and lip height, and complete cleft measurements were shorter than those for incomplete clefts. There were no significant differences in vermilion area, lip width, vermilion height, and Cupid's bow width. CONCLUSIONS: The outcome analysis showed that vermilion height reduction and modifications could be made for both complete and incomplete groups. The complete cleft lip has been found to have a significantly shorter lateral lip length and lip height, reflecting a more severe anatomical soft tissue deficiency in this group. A long-term outcome anthropometric point study may further verify the results of this surgical technique.

A miRNA signature in endothelial cell-derived extracellular vesicles in tumor-bearing mice.

Extracellular vesicles (EVs) play important roles in tumor progression by altering immune surveillance, promoting vascular dysfunction, and priming distant sites for organotropic metastases. The miRNA expression patterns in circulating EVs are important diagnostic tools in cancer. However, multiple cell types within the tumor microenvironment (TME) including cancer cells and stromal cells (e.g. immune cells, fibroblasts, and endothelial cells, ECs) contribute to the pool of circulating EVs. Because EVs of different cellular origins have different functional properties, auditing the cargo derived from cell type-specific EVs in the TME is essential. Here, we demonstrate that a murine EC lineage-tracing model (Cdh5-CreERT2:ZSGreenl/s/l mice) can be used to isolate EC-derived extracellular vesicles (EC-EVs). We further show that purified ZSGreen+ EVs express expected EV markers, they are transferable to multiple recipient cells, and circulating EC-EVs from tumor-bearing mice harbor elevated levels of specific miRNAs (e.g. miR-30c, miR-126, miR-146a, and miR-125b) compared to non tumor-bearing counterparts. These results suggest that, in the tumor setting, ECs may systemically direct the function of heterotypic cell types either in the circulation or in different organ micro-environments via the cargo contained within their EVs.

Cell density regulates in vitro activation of heart valve interstitial cells.

BACKGROUND: Valve interstitial cells, the most prominent cell type in the heart valve, are activated and express α-smooth muscle actin in valve repair and in diseased valves. We hypothesize that cell density, time in culture, and the establishment of cell-cell contacts may be involved in regulating valve interstitial cell activation in vitro. METHODS: To study cell density, valve interstitial cells were plated at passages 3 to 5, at a density of 17,000 cells/22 × 22 mm(2) coverslip, and grown for 1, 2, 4, 7, and 10 days. Valve interstitial cells were stained for α-smooth muscle actin and viewed under confocal microscopy to characterize the intensity of staining. To study time in culture, valve interstitial cells were plated at a 10-fold higher density to achieve similar growth densities over a shorter time period compared with valve interstitial cells plated at low density. α-Smooth muscle actin staining was compared at the same time points between those plated at high and low densities. To confirm valve interstitial cell activation as indicated by α-smooth muscle actin staining, valve interstitial cells were stained for cofilin at days 2, 5, 8, and 14 days postplating. To study the association of transforming growth factor ß with valve interstitial cell activation with respect to cell density, valve interstitial cells were stained for α-smooth muscle actin and transforming growth factor ß at 2, 4, 6, and 8 days postplating. To study the activation of the transforming growth factor ß signaling pathway, valve interstitial cells were stained for pSmad2/3 at days 2, 4, 6, 8, 10, and 12 days postplating. To study cell contacts and activation, subconfluent and confluent cultures of valve interstitial cells were stained for ß-catenin, N-cadherin, and α-smooth muscle actin. Also, whole-cell lysates of subconfluent and confluent valve interstitial cell cultures were probed by Western blot analysis for phospho-ß-catenin at Ser33/37/Thr41, which is the form of ß-catenin targeted for proteosomal degradation. RESULTS: The percentage valve interstitial cells with high-intensity α-smooth muscle actin staining decreases significantly between days 1 and 4, and at confluency, most cells show absent or low-intensity staining, regardless of time in culture. Similar results are obtained with cofilin staining. Transforming growth factor ß and nuclear pSmad2/3 staining in valve interstitial cells decreases concurrently with valve interstitial cell activation as cell density increases. Examining ß-catenin and N-cadherin staining, single valve interstitial cells show no cell-cell contact with strong cytoplasmic staining, with some showing nuclear staining of ß-catenin, while confluent monolayers show strong staining of fully established cell-cell contacts, weak cytoplasmic staining, and absent nuclear staining. The presence of cell-cell contacts is associated with a decreased α-smooth muscle actin. The level of phospho-ß-catenin at Ser33/37/Thr41 is lower in confluent cultures compared with low-density subconfluent valve interstitial cell cultures. CONCLUSION: Cell-cell contacts may inhibit valve interstitial cell activation, while absence of cell-cell contacts may contribute to activation.

Common pathogenic features of atherosclerosis and calcific aortic stenosis: role of transforming growth factor-beta.

Calcific aortic stenosis and atherosclerosis have been investigated separately in experimental in vitro and in vivo studies and in clinical studies. The similarities identified in both diseases suggest that similar pathogenic pathways are involved in both conditions. Most current therapeutic studies are focused on statins. The evidence suggests that statin effects on valves may, in large part, be independent of the lipid lowering effects of the drug. There are several molecules that play significant regulatory roles on the development and progression of valve sclerosis and calcification and on growth and complications of atherosclerotic plaques. The purpose of this review is to discuss the pathogenic features of the two conditions, highlight the important similarities, and then review the data that suggest that transforming growth factor-beta may play a key regulatory role in both diseases and that this is worthy of study as a potential therapeutic target for both conditions.

Transforming growth factor-beta regulates in vitro heart valve repair by activated valve interstitial cells.

The regulation of valve interstitial cell (VIC) function in response to tissue injury and valve disease is not well understood. Because transforming growth factor-beta (TGF-beta) has been implicated in tissue repair, we tested the hypothesis that TGF-beta is a regulator of VIC activation and associated cell responses that occur during early repair processes. We used a well-characterized wound model that was created by mechanical denudation of a confluent VIC monolayer to study activation and repair 24 hours after wounding. VIC activation was demonstrated by immunofluorescent localization of alpha-smooth muscle actin (alpha-SMA), and alpha-SMA mRNA levels were quantified by real-time polymerase chain reaction. Proliferation and apoptosis were quantified by bromodeoxyuridine staining and terminal deoxynucleotidyl transferase dUTP nick end labeling, respectively. Repair was quantified by measuring VIC extension into the wound, and TGF-beta expression was shown by immunofluorescent localization of intracellular TGF-beta. Compared with nonwounded monolayers, VICs at the wound edge showed alpha-SMA staining, increased alpha-SMA mRNA content, elongation into the wound with stress fibers, proliferation, and apoptosis. VICs at the wound edge also showed increased TGF-beta and pSmad2/3 staining with co-expression of alpha-SMA. Addition of TGF-beta neutralizing antibody to the wound decreased VIC activation, alpha-SMA mRNA content, proliferation, apoptosis, wound closure rate, and stress fibers. Conversely, exogenous addition of TGF-beta to the wound increased VIC activation, proliferation, wound closure rate, and stress fibers. Thus, wounding activates VICs, and TGF-beta signaling modulates VIC response to injury.

The emerging role of valve interstitial cell phenotypes in regulating heart valve pathobiology.

The study of the cellular and molecular pathogenesis of heart valve disease is an emerging area of research made possible by the availability of cultures of valve interstitial cells (VICs) and valve endothelial cells (VECs) and by the design and use of in vitro and in vivo experimental systems that model elements of valve biological and pathobiological activity. VICs are the most common cells in the valve and are distinct from other mesenchymal cell types in other organs. We present a conceptual approach to the investigation of VICs by focusing on VIC phenotype-function relationships. Our review suggests that there are five identifiable phenotypes of VICs that define the current understanding of their cellular and molecular functions. These include embryonic progenitor endothelial/mesenchymal cells, quiescent VICs (qVICs), activated VICs (aVICs), progenitor VICs (pVICs), and osteoblastic VICs (obVICs). Although these may exhibit plasticity and may convert from one form to another, compartmentalizing VIC function into distinct phenotypes is useful in bringing clarity to our understanding of VIC pathobiology. We present a conceptual model that is useful in the design and interpretation of studies on the function of an important phenotype in disease, the activated VIC. We hope this review will inspire members of the investigative pathology community to consider valve pathobiology as an exciting new frontier exploring pathogenesis and discovering new therapeutic targets in cardiovascular diseases.

Human tissue kallikrein expression in the stratum corneum and serum of atopic dermatitis patients.

Human tissue kallikreins are a family of 15 trypsin- or chymotrypsin-like secreted serine proteases (KLK1-KLK15). Many KLKs have been identified in normal stratum corneum (SC) and sweat, and are candidate desquamation-related proteases. We report quantification by enzyme-linked immunosorbent assay (ELISA) of KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and KLK14 in the SC and serum of atopic dermatitis (AD) patients by ELISA, and examine their variation with clinical phenotype, correlation with blood levels of eosinophils, lactate dehydrogenase (LDH) and immunoglobulin E. The overall SC serine protease activities were also measured. In the SC of AD, all KLKs, except KLK11, were significantly elevated. The elevation of chymotrypsin-like KLK7 was predominant, compared with trypsin-like KLKs. The SC overall plasmin- and furin-like activities were significantly elevated, while trypsin- and chymotrypsin-like activities did not differ significantly. In the serum of AD patients, KLK8 was significantly elevated and KLK5 and KLK11 were significantly decreased. However, their serum levels were not modified by corticosteroid topical agents. The alterations of KLK levels in the SC of AD were more pronounced than those in the serum. KLK7 in the serum was significantly correlated with eosinophil counts in the blood of AD patients, while KLK5, KLK8 and KLK11 were significantly correlated with LDH in the serum. In conclusion, we report abnormal kallikrein levels in the SC and the serum of AD patients. KLKs might be involved in skin manifestation and/or focal/systemic inflammatory reactions in AD. Our data may contribute to a better understanding of the pathogenesis of AD.