Reversible secretome and signaling defects in diabetic mesenchymal stem cells from peripheral arterial disease patients.

OBJECTIVE: Regenerative medicine seeks to stall or to reverse the pathologic consequences of chronic diseases. Many people with diabetes have peripheral arterial disease (PAD), which increases their already high risk of major amputation. Cellular therapies are a promising regenerative medicine approach to PAD that can be used to focally inject regenerative cells to endangered tissue beds. Mesenchymal stem cells (MSCs) are known to promote tissue regeneration through stromal support and paracrine stimulation of new blood vessels (angiogenesis). Whereas little is known about human diabetic MSCs (dMSCs), particularly those from patients with PAD, dMSCs have a limited expansion capacity but can be improved with human platelet lysate (PL) supplementation. PL is rich in many growth factors, including epidermal growth factor (EGF), which is known to be important to cell proliferation and survival signaling pathways. We hypothesize that dMSCs have a reversible defect in EGF receptor pathways. The objective of this work was to test this hypothesis using dMSCs from PAD patients. METHODS: The secretome expression of EGF and prominent angiogens was characterized from bone marrow (BM)-derived and adipose tissue-derived (ATD) dMSCs from five patients (six limbs) undergoing major amputation. Western blot was used to characterize the AKT and extracellular signal-regulated protein kinases 1 and 2 expression in dMSCs under standard culture (5% fetal bovine serum plus fibroblast growth factor 2 [FGF2]), 5% human PL, or 5% fetal bovine serum plus EGF. Healthy donor MSCs were control cells. The angiogenic activity of BM- and ATD-dMSCs was tested on human umbilical vein endothelial cells (ECs). Paired t-test, analysis of variance, and Kruskal-Wallis tests were used as appropriate. RESULTS: Both BM- and ATD-dMSCs had typical MSC surface marker expression and similar expansion profiles, and they did not express EGF in their secretome. PL supplementation of dMSCs improved AKT signaling, but they were resistant to FGF2 activation of extracellular signal-regulated protein kinases 1 and 2. EGF supplementation led to similar AKT expression as with PL, but PL had greater phosphorylation of AKT at 30 and 60 minutes. The conditioned media from both BM- and ATD-dMSCs had robust levels of prominent angiogens (vascular endothelial growth factor, monocyte chemoattractant protein 1, hepatocyte growth factor), which stimulated EC proliferation and migration, and the co-culture of dMSCs with ECs led to significantly longer EC sprouts in three-dimensional gel than EC-alone pellets. CONCLUSIONS: PL and EGF supplementation improves AKT expression in dMSCs over that of FGF2, but PL improved pAKT over that of EGF. Thus, PL supplementation strategies may improve AKT signaling, which could be important to MSC survival in cellular therapies. Furthermore, BM- and ATD-dMSCs have similar secretomes and robust in vitro angiogenic activity, which supports pursuing dMSCs from both reservoirs in regenerative medicine strategies.

Disturbed Flow Promotes Arterial Stiffening Through Thrombospondin-1.

BACKGROUND: Arterial stiffness and wall shear stress are powerful determinants of cardiovascular health, and arterial stiffness is associated with increased cardiovascular mortality. Low and oscillatory wall shear stress, termed disturbed flow (d-flow), promotes atherosclerotic arterial remodeling, but the relationship between d-flow and arterial stiffness is not well understood. The objective of this study was to define the role of d-flow on arterial stiffening and discover the relevant signaling pathways by which d-flow stiffens arteries. METHODS: D-flow was induced in the carotid arteries of young and old mice of both sexes. Arterial stiffness was quantified ex vivo with cylindrical biaxial mechanical testing and in vivo from duplex ultrasound and compared with unmanipulated carotid arteries from 80-week-old mice. Gene expression and pathway analysis was performed on endothelial cell-enriched RNA and validated by immunohistochemistry. In vitro testing of signaling pathways was performed under oscillatory and laminar wall shear stress conditions. Human arteries from regions of d-flow and stable flow were tested ex vivo to validate critical results from the animal model. RESULTS: D-flow induced arterial stiffening through collagen deposition after partial carotid ligation, and the degree of stiffening was similar to that of unmanipulated carotid arteries from 80-week-old mice. Intimal gene pathway analyses identified transforming growth factor-ß pathways as having a prominent role in this stiffened arterial response, but this was attributable to thrombospondin-1 (TSP-1) stimulation of profibrotic genes and not changes to transforming growth factor-ß. In vitro and in vivo testing under d-flow conditions identified a possible role for TSP-1 activation of transforming growth factor-ß in the upregulation of these genes. TSP-1 knockout animals had significantly less arterial stiffening in response to d-flow than wild-type carotid arteries. Human arteries exposed to d-flow had similar increases TSP-1 and collagen gene expression as seen in our model. CONCLUSIONS: TSP-1 has a critical role in shear-mediated arterial stiffening that is mediated in part through TSP-1's activation of the profibrotic signaling pathways of transforming growth factor-ß. Molecular targets in this pathway may lead to novel therapies to limit arterial stiffening and the progression of disease in arteries exposed to d-flow.

A novel platelet lysate hydrogel for endothelial cell and mesenchymal stem cell-directed neovascularization.

UNLABELLED: Mesenchymal stem cells (MSC) hold promise in promoting vascular regeneration of ischemic tissue in conditions like critical limb ischemia of the leg. However, this approach has been limited in part by poor cell retention and survival after delivery. New biomaterials offer an opportunity to localize cells to the desired tissue after delivery, but also to improve cell survival after delivery. Here we characterize the mechanical and microstructural properties of a novel hydrogel composed of pooled human platelet lysate (PL) and test its ability to promote MSC angiogenic activity using clinically relevant in vitro and in vivo models. This PL hydrogel had comparable storage and loss modulus and behaved as a viscoelastic solid similar to fibrin hydrogels despite having 1/4-1/10th the fibrin content of standard fibrin gels. Additionally, PL hydrogels enabled sustained release of endogenous PDGF-BB for up to 20days and were resistant to protease degradation. PL hydrogel stimulated pro-angiogenic activity by promoting human MSC growth and invasion in a 3D environment, and enhancing endothelial cell sprouting alone and in co-culture with MSCs. When delivered in vivo, the combination of PL and human MSCs improved local tissue perfusion after 8days compared to controls when assessed with laser Doppler perfusion imaging in a murine model of hind limb ischemia. These results support the use of a PL hydrogel as a scaffold for MSC delivery to promote vascular regeneration. STATEMENT OF SIGNIFICANCE: Innovative strategies for improved retention and viability of mesenchymal stem cells (MSCs) are needed for cellular therapies. Human platelet lysate is a potent serum supplement that improves the expansion of MSCs. Here we characterize our novel PL hydrogel's desirable structural and biologic properties for human MSCs and endothelial cells. PL hydrogel can localize cells for retention in the desired tissue, improves cell viability, and augments MSCs' angiogenic activity. As a result of these unique traits, PL hydrogel is ideally suited to serve as a cell delivery vehicle for MSCs injected into ischemic tissues to promote vascular regeneration, as demonstrated here in a murine model of hindlimb ischemia.

Laparoscopic vs open right hepatectomy: a value-based analysis.

BACKGROUND: Current literature lacks sufficient data on outcomes after extensive laparoscopic liver resections. We hypothesized that laparoscopic right hepatectomy (LRH) is associated with better clinical outcomes and less overall hospital costs than open right hepatectomy (ORH), supporting the notion that major laparoscopic hepatic resections carry increased value when compared with the open approach. STUDY DESIGN: We reviewed medical records of all patients at our institution who underwent elective LRH (n = 48) or ORH (n = 57) from May 16, 2008 to March 1, 2012. Patient demographics, preoperative comorbidities, operative details, and postoperative outcomes were compared between the 2 groups. Hospital billing data were collected for each case to determine the average hospital costs per case. RESULTS: Average operative duration, estimated blood loss, intravenous fluid resuscitation requirements, high-grade postoperative complications, the need for postoperative admission to the ICU, and hospital length of stay were significantly less within the LRH cohort. Thirty-day mortality and readmission rates were equivalent between the 2 groups. Despite higher operative costs for LRH ($16,605 vs $10,411, p < 0.001), total postoperative costs were significantly less ($9,075 for LRH vs $16,341 for ORH, p < 0.001), resulting in equivalent overall costs ($25,679 for LRH vs $26,751 for ORH, p = 0.65). CONCLUSIONS: Although overall costs between LRH and ORH are equivalent, clinical outcomes after LRH are comparable to those after ORH, supporting the value of laparoscopy in extensive right hepatic resections. Efforts to reduce operative costs of LRH, while maintaining optimal patient outcomes, should be the focus of surgeons and hospitals moving forward.

Implicaciones clínicas y quirúrgicas de las variaciones anatómicas vasculares del riñón / Clinical and surgical implications of the anatomic variations of the renal vessels / Implicações clínicas cirúrgicas das variações anatômicas vasculares do rim

Introducción: Conocer y diagnosticar las variaciones más frecuentes de la vasculatura renal es de gran importancia para la planificación de la nefrectomía laparoscópica en el donante y para la reconstrucción vascular en el trasplante renal. De igual forma, considerar las variaciones vasculares -especialmente las del sistema venoso- es indispensable en reconstrucción vascular debido a la gran proporción de variaciones venosas asociadas a aneurismas de la aorta abdominal; además, es ideal en el estudio de condiciones clínicas tales como el síndrome de congestión pélvica y la hematuria. Metodología: Se trata de una revisión de la bibliografía sobre la proporción, diagnóstico, procedimientos quirúrgicos y síndromes clínicos asociados a las variaciones de la vasculatura renal, basada en el material encontrado con la siguiente estrategia de búsqueda: "Renal Artery/abnormalities"[Mesh] OR Renal Veins/abnormalities"[Mesh] AND "surgery"[Mesh] OR "transplantation"[Mesh] OR "radiography"[Mesh] "Kidney Pelvis/abnormalities"[Mesh] AND "Kidney Pelvis/blood supply"[Mesh]. Esta estrategia se modificó de acuerdo con las bases de datos: MEDLINE/PubMed, MEDLINE OVID, SCIENCEDIRECT, HINARI y LILACS. Desarrollo: Se revisó el origen y los tipos más frecuentes de variaciones de la vasculatura renal. Se investigó sobre las implicaciones quirúrgicas y los síndromes clínicos asociados.

Introduction: Precise knowledge of the most frequent types of renal vascular anatomical variations, as well as the adequate clinical evaluation is of great importance during laparoscopic donor nephrectomy and during renal vascular reconstruction using kidney allografts with multiple vessels. Equally important is to consider the venous anatomical variations during abdominal vascular reconstruction, particularly when performing an abdominal aortic aneurysm repair, because of the outstanding proportion of renal vascular variations that are associated with this pathology. In addition, it is ideal to think carefully about these variations when a pelvic congestion syndrome or hematuria diagnosis is encountered. Materials and methods: This paper reviews the incidence, diagnosis, surgical procedures, and clinical syndromes associated with renal anatomical vascular variations. We conducted this review taking into account the following Mesh terms: "Renal Artery/abnormalities"[Mesh] OR Renal Veins/abnormalities"[Mesh] AND "surgery"[Mesh] OR "ransplantation"[Mesh] OR "radiography"[Mesh] "Kidney Pelvis/abnormalities"[Mesh] AND "Kidney Pelvis/blood supply"[Mesh]. These terms were adapted with each of the database that was consulted: MEDLINE/PubMed, MEDLINE OVID, SCIENCEDIRECT, HINARI and LILACS. Development: The source and the most frequent types of the vascular anomalies of the kidney were reviewed. We investigated about the associated clinical syndromes and the surgical consequences in kidney transplant.

Introdução. Conhecer e diagnosticar as variações mais freqüentes da vasculatura renal é de grande importância para a planificação da nefrectomia laparoscópica no doador e para a reconstrução vascular no transplante renal. De igual forma, considerar as variações vasculares -especialmente as do sistema venoso- é indispensável em reconstrução vascular devido à grande proporção de variações venosas associadas a aneurismas da aorta abdominal; além disso, é ideal no estudo de condições clínicas tais como a síndrome de congestão pélvica e a hematúria. Metodologia. Trata-se de uma revisão da bibliografia sobre a proporção, diagnóstico, procedimentos cirúrgicos e síndromes clínicas associadas às variações da vasculatura renal, baseada no material encontrado com a seguinte estratégia de procura: "Renal Artery/abnormalities"[Mesh] OR Renal Veins/abnormalities"[Mesh] AND "surgery"[Mesh] OR "transplantation"[Mesh] OR "radiography"[Mesh] "Kidney Pelvis/ abnormalities"[Mesh] AND "Kidney Pelvis/blood supply"[Mesh]. Esta estratégia se modificou de acordo com as bases de dados: MEDLINE/PubMed, MEDLINE OVID, SCIENCEDIRECT, HINARI y LILACS. Desenvolvimento. Se revisou a origem e os tipos mais freqüentes de variações da vasculatura renal. Se investigou sobre as implicações cirúrgicas e as síndromes clínicas associadas.