A thrombospondin-dependent pathway for a protective ER stress response.

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 function for Thbs as ER-resident effectors of an adaptive ER stress response. Thbs4 cardiac-specific transgenic mice were protected from myocardial injury, whereas 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. Thbs bind the ER lumenal domain of activating transcription factor 6α (Atf6α) to promote its nuclear shuttling. Thbs4(-/-) mice showed blunted activation of Atf6α and other ER stress-response factors with injury, and Thbs4-mediated protection was lost upon Atf6α deletion. Hence, Thbs can function inside the cell during disease remodeling to augment ER function and protect through a mechanism involving regulation of Atf6α.

Activating PIK3CA alleles and lymphangiogenic phenotype of lymphatic endothelial cells isolated from lymphatic malformations.

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.

Development of the mechanical properties of engineered skin substitutes after grafting to full-thickness wounds.

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.

Morphogenesis of chimeric hair follicles in engineered skin substitutes with human keratinocytes and murine dermal papilla cells.

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 ß-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.

Assessment of replication rates of human keratinocytes in engineered skin substitutes grafted to athymic mice.

Stable closure of skin wounds with engineered skin substitutes (ESS) requires indefinite mitotic capacity to generate the epidermis. To evaluate whether keratinocytes in ESS exhibit the stem cell phenotype of label retention, ESS (n = 6-9/group) were pulsed with 5-bromo-2'-deoxyuridine (BrdU) in vitro, and after grafting to athymic mice (n = 3-6/group). Pulse and immediate chase in vitro labeled virtually all basal keratinocytes at day 8, with label uptake decreasing until day 22. Label retention in serial chase decreased more rapidly from day 8 to day 22, with a reorganization of BrdU-positive cells into clusters. Similarly, serial chase of labeled basal keratinocytes in vivo decreased sharply from day 20 to day 48 after grafting. Label uptake was assessed by immediate chases of basal keratinocytes, and decreased gradually to day 126, while total labeled cells remained relatively unchanged. These results demonstrate differential rates of label uptake and retention in basal keratinocytes of ESS in vitro and in vivo, and a proliferative phenotype with potential for long-term replication in the absence of hair follicles. Regulation of a proliferative phenotype in keratinocytes of ESS may improve the biological homology of tissue-engineered skin to natural skin, and contribute to more rapid and stable wound healing.

3,4-Methylenedioxy-N-methamphetamine (ecstasy) promotes the survival of fetal dopamine neurons in culture.

The current study examined whether modest concentrations of MDMA could increase the survival and/or neurite outgrowth of fetal midbrain dopamine (DA) neurons in vitro since increased DA neurite outgrowth has been previously observed in vivo from prenatal exposure. MDMA concentrations in fetal brain were quantified to determine relevant in vivo concentrations to employ in vitro. A dose response study in vitro demonstrated that MDMA, at concentrations observed in vivo, resulted in increased, DA-specific, neuron survival. Higher doses resulted in non-specific neurotoxicity. MDMA application immediately after culture establishment resulted in greater survival than delayed application, however both were superior to control. MDMA significantly increased the expression of the slc6a3 gene (dopamine transporter; DAT) in culture. Co-application of the DAT reuptake inhibitor methylphenidate (MPH) with MDMA attenuated this effect. Progressive reductions in MPH concentrations restored the MDMA-induced survival effect. This suggests that MDMA's action at DAT mediates the survival effect. Neurite density per neuron was unaffected by MDMA in vitro suggesting that MDMA promotes DA neuron survival but not neurite outgrowth in culture. Finally, animals prenatally exposed to MDMA and examined on postnatal day 35 showed an increase in tyrosine hydroxylase-positive (TH+) neurons in the substantia nigra but not in the ventral tegmental area. These data suggest that during development, MDMA can increase the survival of DA neurons through its action at its transporter. Understanding how MDMA increases DA neuron survival may provide insight into normal DA neuron loss during development.

Dexamethasone exacerbates cytotoxic chemotherapy induced lethargy and weight loss in female tumor free mice.

Cytotoxic chemotherapy can induce a systemic inflammatory response which has been proposed to be an underlying mechanism of cancer treatment related fatigue. Dexamethasone, a synthetic glucocorticoid that has potent anti-inflammatory effects, is incorporated into chemotherapy regimens to prevent chemotherapy-induced nausea and vomiting (CINV). The purpose of this study was to determine whether by suppressing cytotoxic chemotherapy-induced inflammation, dexamethasone could ameliorate chemotherapy induced fatigue/lethargy in tumor free mice. The effect of dexamethasone (DEX) on Cytoxan-Adriamycin (CA)-induced inflammation was assessed by measuring circulating levels of IL-1ß, TNF-α, IL-6, GCSF, KC, and MCP-1 twenty-four-hours post CA injection. Decline in voluntary wheel running activity (VWRA) from baseline (used as a proxy for fatigue/lethargy), body weight and composition, and food intake were monitored in mice administered four cycles of CA every two weeks with or without DEX. CA increased circulating levels of IL-6, GCSF, KC, and MCP-1 and caused a rapid decline in VWRA and body weight immediately following CA-injection. Although the acute CA-induced decline in VWRA and body weight was not evident in mice administered CA + DEX, DEX alone had a suppressive effect on VWRA, and body weight continued to decline in mice administered both CA and DEX while it returned to baseline in CA-treated mice. CA or DEX alone had no long term impact on VWRA but DEX exacerbated lethargy and weight loss in CA-treated mice. Despite dampening the systemic inflammatory response to chemotherapy, dexamethasone failed to ameliorate acute or long term chemotherapy related fatigue/lethargy. Our pre-clinical findings suggest that supportive therapies like dexamethasone used to acutely control nausea and vomiting in cancer patients may actually contribute to overall symptom burden in cancer patients.

Impaired Cognitive Performance in Endothelial Nitric Oxide Synthase Knockout Mice After Ischemic Stroke: A Pilot Study.

OBJECTIVES: Cognitive dysfunction and dementia are common following ischemic stroke. Endothelial nitric oxide synthase (eNOS) has been found to play an important role in neurologic function and cognition. The purpose of the present study was to assess the specific role of eNOS in cognitive performance after stroke. DESIGN: Male wild-type and mice lacking eNOS (eNOS) underwent middle cerebral artery occlusion or sham-surgery. Primary outcomes were repeated measures of neurologic score, limb asymmetry, sensory/motor function, and spatial memory/learning assessed at intervals up to 28 days postsurgery. Group differences in brain microglia activation and infiltration and levels of interferon-gamma were examined. RESULTS: There was no genotype × surgery interaction effect on the pattern of change in neurologic score, limb asymmetry, or sensory motor function across the 28 days postsurgery. In the Morris water maze, eNOS-/- middle cerebral artery occlusion mice displayed learning and memory deficits not evident in wild-type middle cerebral artery occlusion mice. Poorer spatial memory and learning in eNOS-/- middle cerebral artery occlusion mice was associated with a reduction in the number of activated microglia in the striatum on the lesion side and decreased brain tissue levels of interferon-gamma. CONCLUSIONS: This study's data support a role for eNOS in cognitive performance after stroke. This finding may lead to the development of novel interventions to treat poststroke cognitive deficits.

Characterization of hair follicle development in engineered skin substitutes.

Generation of skin appendages in engineered skin substitutes has been limited by lack of trichogenic potency in cultured postnatal cells. To investigate the feasibility and the limitation of hair regeneration, engineered skin substitutes were prepared with chimeric populations of cultured human keratinocytes from neonatal foreskins and cultured murine dermal papilla cells from adult GFP transgenic mice and grafted orthotopically to full-thickness wounds on athymic mice. Non-cultured dissociated neonatal murine-only skin cells, or cultured human-only skin keratinocytes and fibroblasts without dermal papilla cells served as positive and negative controls respectively. In this study, neonatal murine-only skin substitutes formed external hairs and sebaceous glands, chimeric skin substitutes formed pigmented hairs without sebaceous glands, and human-only skin substitutes formed no follicles or glands. Although chimeric hair cannot erupt readily, removal of upper skin layer exposed keratinized hair shafts at the skin surface. Development of incomplete pilosebaceous units in chimeric hair corresponded with upregulation of hair-related genes, LEF1 and WNT10B, and downregulation of a marker of sebaceous glands, Steroyl-CoA desaturase. Transepidermal water loss was normal in all conditions. This study demonstrated that while sebaceous glands may be involved in hair eruption, they are not required for hair development in engineered skin substitutes.