SUMMARY Thrombospondin (Thbs) proteins are induced in sites of tissue damage or active remodeling. The endoplasmic reticulum (ER) stress response is also prominently induced with disease where it regulates protein production and resolution of misfolded proteins. Here we describe a novel function for Thbs’ as ER resident effectors of an adaptive ER stress response. Thbs4 cardiac-specific transgenic mice were protected from myocardial injury while Thbs4−/− mice were sensitized to cardiac maladaptation. Thbs induction produced a unique profile of adaptive ER stress response factors and expansion of the ER and downstream vesicles. The type-3 repeat domain in Thbs’ bind the ER luminal domain of activating transcription factor 6α (Atf6α) to promote its nuclear shuttling. Thbs4−/−mice failed to show activation of Atf6α and other ER stress response factors with injury, and Thbs4-mediated protection was lost when Atf6α was deleted. Hence, Thbs’ can function inside the cell during disease/remodeling to augment ER function and protect through a mechanism involving regulation of Atf6α.

Lymphatic malformations (LMs) are developmental anomalies of the lymphatic system associated with the dysmorphogenesis of vascular channels lined by lymphatic endothelial cells (LECs). Seeking to identify intrinsic defects in affected LECs, cells were isolated from malformation tissue or fluid on the basis of CD31 and podoplanin (PDPN) expression. LECs from five unrelated LM lesions were characterized, including cells derived from one patient previously diagnosed with CLOVES. CLOVES-related LECs carried a known, activating mutation in PIK3CA (p.H1047L), confirmed by direct sequencing. Activating PIK3CA mutations (p.E542K and p.E545A) were identified in lesion-derived cells from the other four patients, also by direct sequencing. The five LM-LEC cultures shared a lymphangiogenic phenotype distinguished by PI3K/AKT activation, enhanced sprouting efficiency, elevated VEGF-C expression and COX2 expression, shorter doubling times and reduced expression of angiopoietin 2 and CXCR4. Nine additional LM-LEC populations and 12 of 15 archived LM tissue samples were shown to bear common PIK3CA variants by allele-specific PCR. The activation of a central growth/survival pathway (PI3K/AKT) represents a feasible target for the non-invasive treatment of LMs bearing in mind that background genetics may individualize lesions and influence treatments.

Engineered skin substitutes (ESS) have been used successfully to treat life-threatening burns, but lack cutaneous appendages. To address this deficiency, dermal constructs were prepared using collagen-glycosaminoglycan scaffolds populated with murine dermal papilla cells expressing green fluorescent protein (mDPC-GFP), human dermal papilla cells (hDPC) and/or human fibroblasts (hF). Subsequently, human epidermal keratinocytes (hK) or hK genetically modified to overexpress stabilized b-catenin (hK') were used to prepare ESS epithelium. After 10 days incubation at air-liquid interface, ESS were grafted to athymic mice and were evaluated for 6 weeks. Neofollicles were observed in ESS containing mDPC-GFP, but not hDPC or hF, independent of whether or not the hK were genetically modified. Based on detection of GFP fluorescence, mDPC were localized to the dermal papillae of the well-defined follicular structures of grafted ESS. In addition, statistically significant increases in LEF1, WNT10A and WNT10B were found in ESS with neofollicles. These results demonstrate a model for generation of chimeric hair in ESS.

Engineered skin substitutes (ESSs) have been reported to close full-thickness burn wounds but are subject to loss from mechanical shear due to their deficiencies in tensile strength and elasticity. Hypothetically, if the mechanical properties of ESS matched those of native skin, losses due to shear or fracture could be reduced. To consider modifications of the composition of ESS to improve homology with native skin, biomechanical analyses of the current composition of ESS were performed. ESSs consist of a degradable biopolymer scaffold of type I collagen and chondroitin-sulfate (CGS) that is populated sequentially with cultured human dermal fibroblasts (hF) and epidermal keratinocytes (hK). In the current study, the hydrated biopolymer scaffold (CGS), the scaffold populated with hF dermal skin substitute (DSS), or the complete ESS were evaluated mechanically for linear stiffness (N/mm), ultimate tensile load at failure (N), maximum extension at failure (mm), and energy absorbed up to the point of failure (N-mm). These biomechanical end points were also used to evaluate ESS at six weeks after grafting to full-thickness skin wounds in athymic mice and compared to murine autograft or excised murine skin. The data showed statistically significant differences (p <0.05) between ESS in vitro and after grafting for all four structural properties. Grafted ESS differed statistically from murine autograft with respect to maximum extension at failure, and from intact murine skin with respect to linear stiffness and maximum extension. These results demonstrate rapid changes in mechanical properties of ESS after grafting that are comparable to murine autograft. These values provide instruction for improvement of the biomechanical properties of ESS in vitro that may reduce clinical morbidity from graft loss.