Leptin enhances endothelial cell differentiation and angiogenesis in murine embryonic stem cells.

The metabolic regulation of leptin and its angiogenic effects have been well characterized in adult mammals. However, the role of leptin in the differentiation of embryonic stem cells (ESCs) to endothelial cells (ECs) has not been characterized. We hypothesized that leptin enhances the generation of ECs derived from ESCs and, in this way, promotes angiogenesis in embryonic vessels. To address this hypothesis, we utilized an in vitro model consisting of murine ESCs-derived embryoid bodies (EBs). Vascular density, EC and angiogenesis markers as well as phosphorylation levels of signal transducer and activator of transcription 3 (pSTAT3) were investigated in leptin-treated EBs and in untreated EBs as controls. ESC-derived ECs were isolated by magnetic sorting based on the expression of platelet endothelial cell adhesion molecule (PECAM-1/CD31). Significant upregulation of EC and angiogenic markers as well as higher vessel density were found in leptin-treated EBs compared to controls. CD31 positive enriched cells derived from leptin-treated EBs had improved proliferation and survival rate and showed higher levels of pSTAT3. These results suggested that leptin promotes EC differentiation and angiogenesis in mouse EBs and that janus tyrosine kinase (JAK)/STAT pathway can play a role in this biological process. Leptin-mediated EC differentiation and angiogenesis in ESCs can be a useful application towards regenerative medicine and tissue engineering.

Pre-hospital hypothermia is not associated with increased survival after traumatic brain injury.

BACKGROUND: Conclusions from in vivo and in vitro studies suggest hypothermia may be protective in traumatic brain injury (TBI). Few studies evaluated the effect of admission temperature on outcomes. The purpose of this study is to examine the relationship between admission hypothermia and mortality in patients with isolated, blunt, moderate to severe TBI. METHODS: The Los Angeles Trauma Database was queried for all patients ≥ 14 y of age with isolated, blunt, moderate to severe TBI (head abbreviated injury score (AIS) ≥ 3, all other <3), admitted between 2005 and 2009. The study population was then stratified into two groups by admission temperature: hypothermic (≤ 35°C) and normothermic (>35°C). Demographic characteristics and outcomes were compared between groups. Logistic regression analysis was used to determine the relationship between admission hypothermia and mortality. RESULTS: A total of 1834 patients were analyzed and then stratified into two groups: hypothermic (n = 44) and normothermic (n = 1790). There was a significant difference noted in overall mortality (25% versus 7%), with the hypothermic group being four times more likely to succumb to their injuries. After adjusting for confounding factors, admission hypothermia was independently associated with increased mortality (AOR 2.5; 95% CI 1.1-6.3; P = 0.04). CONCLUSIONS: Although in-vivo and in-vitro studies demonstrate induced hypothermia may be protective in TBI, our study demonstrates that admission hypothermia was associated with increased mortality in isolated, blunt, moderate to severe TBI. Further prospective research is needed to elucidate the role of thermoregulation in patients sustaining TBI.

Characterization of microvascular endothelial cells isolated from the dermis of adult mouse tails.

Dermal microvascular endothelial cells (DMECs) play an important role in physiological and pathophysiological processes such as wound healing, cell differentiation, antigen-presentation, inflammation, tumor metastasis, and diabetes. The study of these processes requires a suitable and accessible in vitro model, such as murine DMECs (mDMECs). However, since these cells are difficult to isolate and propagate, some of their properties are not fully characterized. We isolated these cells from C57BL/6J adult mouse tail skin and purified them using magnetic sorting. Then, we tested several culture conditions and oxygen concentrations for mDMEC growth and propagation. After obtaining optimal culture conditions, we characterized the expression of EC markers and compared such expression with an established murine microvascular EC line (EOMA). Our results indicate that mDMECs isolated from mouse tails expressed most of the characteristic EC markers such as von Willebrand Factor (vWF), CD31, Tie1, Tie2, ANGPT1, ANGPT2, FLK-1, FLT-1, and VEGF-A. Further characterization demonstrated that these cells also expressed proteins involved in organogenesis such as bone morphogenetic proteins-2, -4 (BMP-2/-4), and their receptor (BMPR1A). Surprisingly, higher expression of vWF, ANGPT1, and BMP-2 was observed in mDMECs compared to EOMA cells. For mDMEC in vitro propagation, we found a twofold increase in cell proliferation in cells that grew at 1% O(2) compared to those cells that grew at standard 20% O(2.) Therefore, the method described herein for mDMECs isolation and propagation allowed us to analyze in more detail their biological properties that can be relevant for the study of pathological processes using mouse models.

Pre-hospital intubation is associated with increased mortality after traumatic brain injury.

BACKGROUND: Early endotracheal intubation in patients sustaining moderate to severe traumatic brain injury (TBI) is considered the standard of care. Yet the benefit of pre-hospital intubation (PHI) in patients with TBI is questionable. The purpose of this study was to investigate the relationship between pre-hospital endotracheal intubation and mortality in patients with isolated moderate to severe TBI. METHODS: The Los Angeles County Trauma System Database was queried for all patients > 14 y of age with isolated moderate to severe TBI admitted between 2005 and 2009. The study population was then stratified into two groups: those patients requiring intubation in the field (PHI group) and those patients with delayed airway management (No-PHI group). Demographic characteristics and outcomes were compared between groups. Multivariate analysis was used to determine the relationship between pre-hospital endotracheal intubation and mortality. RESULTS: A total of 2549 patients were analyzed and then stratified into the two groups: PHI and No-PHI. There was a significant difference noted in overall mortality (90.2% versus 12.4%), with the PHI group being more likely to succumb to their injuries. After adjusting for possible confounding factors, multivariable logistic regression analysis demonstrated that PHI was independently associated with increased mortality (AOR 5, 95% CI: 1.7-13.7, P = 0.004). CONCLUSIONS: Pre-hospital endotracheal intubation in isolated, moderate to severe TBI patients is associated with a nearly 5-fold increase in mortality. Further prospective studies are required to establish guidelines for optimal pre-hospital management of this critically injured patient population.

Silicone Migration and Late Hematoma following Silicone Implant Rupture: Case Report and Literature Review.

Distant silicone migration and late postoperative hematoma are rare but serious complications following breast implant rupture. This study describes a case report of both these complications occurring in the same patient. After a review of pertinent literature, the authors found 19 other case reports (20 total patients) with distant silicone migration following breast implant rupture. Median age at the time of presentation was 48 years (range, 21-76), and median time between initial breast augmentation and presentation with silicone migration was 10 years (range, 1-30 years). Sites of migrated silicone included arm/forearm (n = 11), thoracic cavity (n = 4), abdominal wall (n = 3), legs (n = 2), and back (n = 1). A total of 67% of patients had documented trauma to the chest before presentation. Our study highlights the need to consider distant silicone migration in the differential diagnosis when extracapsular implant rupture is suspected.

Effect of a short-term diet and exercise intervention on oxidative stress, inflammation, MMP-9, and monocyte chemotactic activity in men with metabolic syndrome factors.

The present study was designed to examine the effects of lifestyle modification on key contributing factors to atherogenesis, including oxidative stress, inflammation, chemotaxis, and cell adhesion. Obese men (n = 31), 15 of whom had metabolic syndrome, were placed on a high-fiber, low-fat diet in a 3-wk residential program where food was provided ad libitum and daily aerobic exercise was performed. In each subject, pre- and postintervention fasting blood was drawn for circulating levels of serum lipids, glucose and insulin (for estimation of insulin sensitivity), oxidative stress-generating enzyme myeloperoxidase and marker 8-isoprostaglandin F2alpha, the inflammatory protein C-reactive protein, soluble ICAM-1 as an indicator of endothelial activation, sP-selectin as a marker of platelet activation, the chemokine macrophage inflammatory protein-1alpha, and total matrix metalloproteinase-9. Using subject sera and human aortic endothelial cell culture systems, we measured VCAM-1 cell surface abundance and monocyte chemotactic protein-1, nitric oxide, superoxide, and hydrogen peroxide production in vitro by fluorometric detection. Also determined in vitro was serum-induced, monocyte adhesion and monocyte chemotactic activity. After 3 wk, significant reductions (P < 0.05) in body mass index, all serum lipids and lipid ratios, fasting glucose, insulin, homeostasis model assessment for insulin resistance, myeloperoxidase, 8-isoprostaglandin F2alpha, C-reactive protein, soluble ICAM-1, soluble P-selectin, macrophage inflammatory protein-1alpha, and matrix metalloproteinase-9 were noted. In vitro, serum-stimulated cellular VCAM-1 expression, monocyte chemotactic protein-1 production, and fluorometric detection of superoxide and hydrogen peroxide production decreased, whereas a concomitant increase in NO production was noted (all P < 0.01). Additionally, both monocyte adhesion (P < 0.05) and MCA (P < 0.01) decreased. Nine of 15 were no longer positive for metabolic syndrome postintervention. Intensive lifestyle modification may ameliorate novel coronary artery disease risk factors in men with metabolic syndrome factors before reversal of obesity.

Bone morphogenetic protein-2/-4 upregulation promoted by endothelial cells in coculture enhances mouse embryoid body differentiation.

Endothelial cells (ECs) provide inductive signals for cell differentiation in vivo. However, it is unknown if these cells promote such differentiation in vitro and the signals involved. We investigated whether ECs are able to enhance the differentiation of the three germ layers and the underlying mechanisms. We established a coculture system of mouse embryoid bodies (EBs) and ECs. Then, we analyzed the expression of markers representative of the three germ layers, such as PDX-1, proinsulin, insulin1 (endoderm), nestin, neurofilament light (ectoderm), CD31, cardiotin, and cardiac troponin I (mesoderm) in EBs cultured alone (controls) or with ECs. A significant increase of these markers was observed in EBs cocultured with ECs compared to controls. The cocultured EBs also exhibited more robust vascular networks similar to those EBs treated with bone morphogenetic protein-2 or -4 (BMP-2 or -4). Therefore, the role of these peptides in the differentiation was investigated. We found a significant upregulation of BMP-2/-4 and BMP receptor 1A in EBs treated with EC conditioned medium (EC-CM) at early or middle stages of EB development. Recombinant human BMP-2 and BMP-4 exerted similar effects than EC-CM in the expression of BMPs or in the upregulation of the three germ layer specific markers. BMP-2/-4 antagonists, such as noggin and chordin-like-1, respectively inhibited the EC-CM inductive effects. These results demonstrate that ECs enhance the differentiation in vitro of cells that derived from the three germ layers and that BMP-2/-4 play a central role in this process.

Endothelial cells in co-culture enhance embryonic stem cell differentiation to pancreatic progenitors and insulin-producing cells through BMP signaling.

Endothelial cells (ECs) represent the major component of the embryonic pancreatic niche and play a key role in the differentiation of insulin-producing ß cells in vivo. However, it is unknown if ECs promote such differentiation in vitro. We investigated whether interaction of ECs with mouse embryoid bodies (EBs) in culture promotes differentiation of pancreatic progenitors and insulin-producing cells and the mechanisms involved. We developed a co-culture system of mouse EBs and human microvascular ECs (HMECs). An increase in the expression of the pancreatic markers PDX-1, Ngn3, Nkx6.1, proinsulin, GLUT-2, and Ptf1a was observed at the interface between EBs and ECs (EB-EC). No expression of these markers was found at the periphery of EBs cultured without ECs or those co-cultured with mouse embryonic fibroblasts (MEFs). At EB-EC interface, proinsulin and Nkx6.1 positive cells co-expressed phospho-Smad1/5/8 (pSmad1/5/8). Therefore, EBs were treated with HMEC conditioned media (HMEC-CM) suspecting soluble factors involved in bone morphogenetic protein (BMP) pathway activation. Upregulation of PDX-1, Ngn3, Nkx6.1, insulin-1, insulin-2, amylin, SUR1, GKS, and amylase as well as down-regulation of SST were detected in treated EBs. In addition, higher expression of BMP-2/-4 and their receptor (BMPR1A) were also found in these EBs. Recombinant human BMP-2 (rhBMP-2) mimicked the effects of the HMEC-CM on EBs. Noggin (NOG), a BMP antagonist, partially inhibited these effects. These results indicate that the differentiation of EBs to pancreatic progenitors and insulin-producing cells can be enhanced by ECs in vitro and that BMP pathway activation is central to this process.