Introduction of ligated vessels promote the retention and regeneration of free fat: constructing a fat flap in tissue engineering chamber.

Background: Breast reconstruction with fat grafting has an unstable retention rate due to insufficient revascularization. Tissue Engineering Chamber (TEC) model can promote tissue regeneration in the chamber by introducing ligated vessels around the tissue. We introduced ligated vessels with free fat graft to investigate the retention rate and revascularization of grafted fat that in TEC model.Methods: SD rats (n=24) was divided into 3 groups randomly. Group A: Standard TEC model was constructed; Group B: the epigastric vessel bundles were dissected from the fat flap and ligated, fat flap was cut into granules and planted into the chamber; Group C: Free fat was planted in the chamber. At week 6, samples in the chamber were harvested.Results: Significant volume increase was observed in group A and B, while the volume decreased in group C (P<0.05). Regeneration morphology could be found according to the histological observation in A and B. Micro CT results showed the ligated vessels into grafted fat sprouting robustly, coordinated with volume changes.Conclusion: Fat grafts in TEC model could not only survive but also regenerate. The combination of fat graft and TEC could fabricate a vascularized fat flap, which was a promising method in breast reconstruction.Abbreviations: VOI: Volumes of Interest; TEC: Tissue Engineering Chamber; CAL: Cell Assisted Lipotransfer.

Female sex and Western-style diet protect mouse resistance arteries during acute oxidative stress.

A Western-style diet (WD; high in fat and carbohydrates) increases vascular oxidative stress. We hypothesized that vascular cells adapt to a WD by developing resilience to oxidative stress. Male and female C57BL/6J mice (4 wk of age) were fed a control diet (CD) or a WD for 16-20 wk. Superior epigastric arteries (SEAs; diameter, ~125 µm) were isolated and pressurized for study. Basal reactive oxygen species production was greatest in SEAs from males fed the WD. During exposure to H2O2 (200 µM, 50 min), propidium iodide staining identified nuclei of disrupted endothelial cells (ECs) and smooth muscle cells (SMCs). For mice fed the CD, death of SMCs (21%) and ECs (6%) was greater (P < 0.05) in SEAs from males than females (9% and 2%, respectively). WD consumption attenuated cell death most effectively in SEAs from males. With no difference at rest, H2O2 increased intracellular Ca2+ concentration ([Ca2+]i) to the greatest extent in SEAs from males, as shown by fura 2 fluorescence. Selective disruption of the endothelium (luminal air bubble) increased [Ca2+]i and SMC death during H2O2 exposure irrespective of sex; the WD reduced both responses most effectively in males. Nonselective transient receptor potential (TRP) channel inhibition (ruthenium red, 5 µM) attenuated the rise of [Ca2+]i, as did selective inhibition of TRP vanilloid type 4 (TRPV4) channels (HC-067047, 1 µM), which also attenuated cell death. In contrast, inhibition of voltage-gated Ca2+ channels (diltiazem, 50 µM) was without effect. Thus, for resistance arteries during acute oxidative stress: 1) ECs are more resilient than (and can protect) SMCs, 2) vessels from females are inherently more resilient than those from males, and 3) a WD increases vascular resilience by diminishing TRPV4 channel-dependent Ca2+ entry.

Advanced age protects resistance arteries of mouse skeletal muscle from oxidative stress through attenuating apoptosis induced by hydrogen peroxide.

KEY POINTS: Vascular oxidative stress increases with advancing age. We hypothesized that resistance vessels develop resilience to oxidative stress to protect functional integrity and tested this hypothesis by exposing isolated pressurized superior epigastric arteries (SEAs) of old and young mice to H2 O2 . H2 O2 -induced death was greater in smooth muscle cells (SMCs) than endothelial cells (ECs) and lower in SEAs from old vs. young mice; the rise in vessel wall [Ca2+ ]i induced by H2 O2 was attenuated with ageing, as was the decline in noradrenergic vasoconstriction; genetic deletion of IL-10 mimicked the effects of advanced age on cell survival. Inhibiting NO synthase or scavenging peroxynitrite reduced SMC death; endothelial denudation or inhibiting gap junctions increased SMC death; delocalization of cytochrome C activated caspases 9 and 3 to induce apoptosis. Vascular cells develop resilience to H2 O2 during ageing by preventing Ca2+ overload and endothelial integrity promotes SMC survival. ABSTRACT: Advanced age is associated with elevated oxidative stress and can protect the endothelium from cell death induced by H2 O2 . Whether such protection occurs for intact vessels or differs between smooth muscle cell (SMC) and endothelial cell (EC) layers is unknown. We tested the hypothesis that ageing protects SMCs and ECs during acute exposure to H2 O2 (200 µm, 50 min). Mouse superior epigastric arteries (SEAs; diameter, ∼150 µm) were isolated and pressurized to 100 cmH2 O at 37˚C. For SEAs from young (4 months) mice, H2 O2 killed 57% of SMCs and 11% of ECs in males vs. 8% and 2%, respectively, in females. Therefore, SEAs from males were studied to resolve the effect of ageing and experimental interventions. For old (24 months) mice, SMC death was reduced to 10% with diminished accumulation of [Ca2+ ]i in the vessel wall during H2 O2 exposure. In young mice, genetic deletion of IL-10 mimicked the protective effect of ageing on cell death and [Ca2+ ]i accumulation. Whereas endothelial denudation or gap junction inhibition (carbenoxolone; 100 µm) increased SMC death, inhibiting NO synthase (l-NAME, 100 µm) or scavenging peroxynitrite (FeTPPS, 5 µm) reduced SMC death along with [Ca2+ ]i . Despite NO toxicity via peroxynitrite formation, endothelial integrity protects SMCs. Caspase inhibition (Z-VAD-FMK, 50 µm) attenuated cell death with immunostaining for annexin V, cytochrome C, and caspases 3 and 9 pointing to induction of intrinsic apoptosis during H2 O2 exposure. We conclude that advanced age reduces Ca2+ influx that triggers apoptosis, thereby promoting resilience of the vascular wall during oxidative stress.

Increased amplitude of inward rectifier K+ currents with advanced age in smooth muscle cells of murine superior epigastric arteries.

Inward rectifier K+ channels (KIR) may contribute to skeletal muscle blood flow regulation and adapt to advanced age. Using mouse abdominal wall superior epigastric arteries (SEAs) from either young (3-6 mo) or old (24-26 mo) male C57BL/6 mice, we investigated whether SEA smooth muscle cells (SMCs) express functional KIR channels and how aging may affect KIR function. Freshly dissected SEAs were either enzymatically dissociated to isolate SMCs for electrophysiological recording (perforated patch) and mRNA expression or used intact for pressure myography. With 5 mM extracellular K+ concentration ([K+]o), exposure of SMCs to the KIR blocker Ba2+ (100 µM) had no significant effect (P > 0.05) on whole cell currents elicited by membrane potentials spanning -120 to -30 mV. Raising [K+]o to 15 mM activated Ba2+-sensitive KIR currents between -120 and -30 mV, which were greater in SMCs from old mice than in SMCs from young mice (P < 0.05). Pressure myography of SEAs revealed that while aging decreased maximum vessel diameter by ~8% (P < 0.05), it had no significant effect on resting diameter, myogenic tone, dilation to 15 mM [K+]o, Ba2+-induced constriction in 5 mM [K+]o, or constriction induced by 15 mM [K+]o in the presence of Ba2+ (P > 0.05). Quantitative RT-PCR revealed SMC expression of KIR2.1 and KIR2.2 mRNA that was not affected by age. Barium-induced constriction of SEAs from young and old mice suggests an integral role for KIR in regulating resting membrane potential and vasomotor tone. Increased functional expression of KIR channels during advanced age may compensate for other age-related changes in SEA function.NEW & NOTEWORTHY Ion channels are integral to blood flow regulation. We found greater functional expression of inward rectifying K+ channels in smooth muscle cells of resistance arteries of mouse skeletal muscle with advanced age. This adaptation to aging may contribute to the maintenance of vasomotor tone and blood flow regulation during exercise.

Expression of BMP-2 in Vascular Endothelial Cells of Recipient May Predict Delayed Graft Function After Renal Transplantation.

BACKGROUND/AIMS: Delayed graft function (DGF) is associated with adverse outcomes after renal transplantation. Bone morphogenetic protein-2 (BMP-2) is involved in both endothelial function and immunological events. We compared expression of BMP-2 in epigastric artery of renal transplant recipients with immediate graft function (IGF) and DGF. METHODS: 79 patients were included in this prospective study. Patients were divided in IGF group (64 patients) and DGF group (15 patients). BMP-2 expression in intima media (BMP2m) and endothelium (BMP2e) of epigastric artery was assessed by immunohistochemistry. RESULTS: Lower intensity of BMP2e staining was recorded in DGF compared to IGF. In DGF patients, 93% had no expression of BMP2e and 7% had 1st grade expression, compared to 45% and 41% in IGF group, respectively (P=0.001) (P<0.001 for no expression and P = 0.015 for 1st grade expression). Patients who had BMP2e staining positive had lower odds for DGF (OR 0.059 [0.007, 0.477]) and this remained significant even after adjustment for donor and recipient variables, cold ischemia time, and immunological matching (OR 0.038 [0.003, 0.492]). CONCLUSIONS: Our results demonstrate that BMP-2 expression in endothelial cells of epigastric arteries may predict development of DGF.

Vascular smooth muscle desensitization in rabbit epigastric and mesenteric arteries during hemorrhagic shock.

The decompensatory phase of hemorrhage (shock) is caused by a poorly defined phenomenon termed vascular hyporeactivity (VHR). VHR may reflect an acute in vivo imbalance in levels of contractile and relaxant stimuli favoring net vascular smooth muscle (VSM) relaxation. Alternatively, VHR may be caused by intrinsic VSM desensitization of contraction resulting from prior exposure to high levels of stimuli that temporarily adjusts cell signaling systems. Net relaxation, but not desensitization, would be expected to resolve rapidly in an artery segment removed from the in vivo shock environment and examined in vitro in a fresh solution. Our aim was to 1) induce shock in rabbits and apply an in vitro mechanical analysis on muscular arteries isolated pre- and postshock to determine whether VHR involves intrinsic VSM desensitization, and 2) identify whether net VSM relaxation induced by nitric oxide and cyclic nucleotide-dependent protein kinase activation in vitro can be sustained for some time after relaxant stimulus washout. The potencies of phenylephrine- and histamine-induced contractions in in vitro epigastric artery removed from rabbits posthemorrhage were decreased by ∼0.3 log units compared with the control contralateral epigastric artery removed prehemorrhage. Moreover, a decrease in KCl-induced tonic, relative to phasic, tension of in vitro mesenteric artery correlated with the degree of shock severity as assessed by rates of lactate and K(+) accumulation. VSM desensitization was also caused by tyramine in vivo and PE in vitro, but not by relaxant agents in vitro. Together, these results support the hypothesis that VHR during hemorrhagic decompensation involves contractile stimulus-induced long-lasting, intrinsic VSM desensitization.

The Protective Effects of Botulinum Toxin A Against Flap Necrosis After Perforator Twisting and Its Underlying Molecular Mechanism in a Rat Model.

PURPOSE: The purpose of this study was to examine our hypotheses that botulinum toxin A (BoTA) protect necrosis of perforator flap from perforator twisting. METHODS: Twenty-four rats were randomly divided into 2 groups. Twelve International Units of BoTA versus 1.2 mL normal saline was injected subdermally 3 days before flap elevation. In each group, bilateral before deep inferior epigastric perforator (DIEP) flaps, 5 × 3 cm in size, were created. The right and left (180 and 360 degrees of perforator twisting) DIEP flaps were separated. At 1 and 3 days postoperatively, skin above the perforator of the DIEP flaps was harvested to examine the degrees of gene expressions. Final survival percentage of flap and histology were assessed at postoperative day 5. RESULTS: The survival percentage of flap was significantly higher in the BoTA group than in the control group at both DIEP flaps after 180 and 360 degrees of perforator twisting at postoperative day 5 (95.23 ± 2.85% vs 91.00 ± 3.77%; P = 0.021 and 91.59 ± 2.87% vs 30.03 ± 6.91%; P < 0.001, respectively).Higher fibroblast density, enhanced epithelial necrosis, and inflammation were noted in the control group than in the BoTA group. In 180 degrees of perforator twisting group, BoTA may augment angiogenesis possibly via nuclear factor-κB-induced destabilization and the nuclear factor-κB/hypoxia-inducible factor 1-α/vascular endothelial growth factor pathway, whereas in the 360 degrees of perforator twisting group, the mechanistic target of rapamycin/hypoxia-inducible factor 1-α/vascular endothelial growth factor pathway may participate in BoTA-induced effective angiogenesis. CONCLUSIONS: We demonstrated that pretreatment with BoTA protects perforator flap caused by perforator at the pathological and molecular level using an experimental rat model.

Well differentiated neuroendocrine tumor of the appendix and low-grade appendiceal mucinous neoplasm presenting as a collision tumor

No disponible

Collagen-induced thrombosis in murine arteries and veins.

Collagen is a powerful thrombotic stimulus that functions by direct and indirect binding to various platelet receptors. A variety of collagen types are known and several (e.g., collagen Types I, III, IV) are found in vascular tissues and are exposed upon disruption of the endothelium or more extensive vessel wall rupture. Some murine models of thrombosis purport to expose collagen to initiate thrombosis, however, the nature and extent of this exposure is not clear. This study was undertaken to place a known collagen-dominated surface into the in vivo arterial or venous circulation as a method for direct study of collagen-induced thrombosis in mice. The epigastric artery was removed from donor mice and a microsuture with attached needle was knotted into one cut end. Anesthetized mice had this needle/suture/small-artery inserted into and out of a 0.5-mm length of the larger carotid artery or femoral vein, leaving the collagen-rich adventitial surface of the epigastric artery intralumenally in the larger vessel. Extensive platelet and fibrin deposition on this surface were in evidence and were quantitated with fluorescence imaging; administration of clopidogrel reduced thrombus development in both arteries and veins. A method was developed to evert the epigastric artery and disrupt the exteriorized endothelium; with the same needle/suture vessel-insertion technique, this surface stimulated significantly less thrombotic response in both arteries and veins, suggesting differential thrombogenesis based on the molecular composition of the induction factor. This new model of thrombosis offers a method for directly assessing the role of collagen-mediated thrombosis in murine arteries and veins.

Adipose derived stem cells protect skin flaps against ischemia-reperfusion injury.

BACKGROUND: Advances in the treatment of ischemia- reperfusion injury have created an opportunity for plastic surgeons to apply these treatments to flaps and implanted tissues. We examined the capability of adipose derived stem cells (ADSCs) to protect tissue against IRI using an extended inferior epigastric artery skin flap as a flap ischemia- reperfusion injury (IRI) model. METHODS: ADSCs were isolated from Lewis rats and cultured in vitro. Twenty- four rats were randomly divided into three groups. Group I was the sham group and did not undergo ischemic insult; rather, the flap was raised and immediately sutured back (non-ischemic control group). Group II (ischemia control) and group III (ADSCs treatment) underwent 3 h of ischemic insult. During reperfusion group III was treated by intravenous application of ADSCs and group II was left untreated. Five days postoperatively, flap survival and perfusion were assessed. Microvessel density was visualized by immunohistochemistry and semi- quantitative real-time polymerase chain reaction addressed differential gene expression. RESULTS: Treatment with ADSCs significantly increased flap survival (p<0.001) and flap perfusion (p<0.001) when compared to the control group II. Microvessel- density in ADSCs treated group was not significantly increased in any group. No significant differences showed the comparison of the experimental group III and the sham operated control group I. ADSCs treatment (Group III) was accompanied by a significantly enhanced expression of pro-angiogenic and pro-inflammatory genes. CONCLUSION: Overall, our study demonstrates that ADSCs treatment significantly enhances skin flap survival in the aftermath of ischemia to an extent that almost equals surgical results without ischemia. This effect is accompanied with a pronounced and significant angiogenic response and an improved blood perfusion.

Involvement of parathyroid hormone-related protein in vascular calcification of chronic haemodialysis patients.

AIMS: To investigate the role of parathyroid hormone-related protein (PTHrP) in vascular calcification of patients with chronic hemodialysis. METHODS: The inferior epigastric arteries were obtained from 23 patients on chronic haemodialysis and 16 patients with renal carcinoma as control. Haematoxylin-eosin staining, elastic fibre staining, Alizarin Red calcium staining and immunohistochemical staining of PTHrP, bone morphogenetic protein-2 (BMP-2), Cbfa1/Runx2 were performed. Real-time polymerase chain reaction (PCR) was used to examine mRNA expressions of PTHrP, BMP-2 and Cbfa1/Runx2. Western blot and real-time PCR were used to detect the effects of PTHrP-siRNA and rh-PTHrP-1-34 on the expressions of PTHrP, BMP-2 and Cbfa1/Runx2 in human aortic smooth muscle cells (HASMC). Alkaline phosphatase (ALP) activities and intracellular calcium content in HASMCs were assessed after treatment with 10 mmol/L ß-glycerol phosphoric acid for 48 h. RESULTS: Vascular calcification was confirmed in 78.2% of patients on chronic haemodialysis, and the expressions of PTHrP, BMP-2 and Cbfa1 in the arteries were significantly upregulated. PTHrP-siRNA could downregulate the expression of PTHrP by 60%, BMP-2 by 25% and Cbfa1 by 25% at 24 h (P < 0.05). Exogenous rh-PTHrP-1-34 could upregulate the expressions of BMP-2 and Cbfa1 by 1.37-fold and 1.46-fold, respectively, at 24 h in a time-independent manner (P < 0.05), which were attenuated by PTHrP-siRNA. Moreover, it could promote intracellular calcium deposition and increase ALP activities, which were partially blocked by PTHrP-siRNA (P < 0.05). CONCLUSIONS: Vascular calcification was common in patients with chronic haemodialysis, to which PTHrP might contribute by activating BMP-2/ Cbfa1 signalling pathway.

Type 1 diabetes increases the expression of proinflammatory cytokines and adhesion molecules in the artery wall of candidate patients for kidney transplantation.

OBJECTIVE: Diabetes may accelerate atheromatosis in uremic patients. Our aim was to assess the influence of type 1 diabetes on the atheromatosis-related inflammation in patients with chronic kidney disease (CKD). RESEARCH DESIGN AND METHODS: We analyzed the expression of proinflammatory cytokines and adhesion molecules in the inferior epigastric artery walls of type 1 diabetic patients with CKD (n = 22) and compared it with nondiabetic uremic patients (n = 92) at the time of kidney transplantation. We evaluated the expression of interleukin (IL)-6, monocyte chemotractant protein (MCP)-1, vascular cell adhesion molecule (VCAM)-1, intercellular adhesion molecule-1, and the activation of nuclear factor-κß p65 (NFkB-p65). Common carotid intima-media thickness (c-IMT) was determined by conventional echography. RESULTS: IL-6, MCP-1, and VCAM-1 proteins were elevated in type 1 diabetic patients compared with nondiabetic subjects (P < 0.05). The nuclear localization of NFkB-p65 was higher in type 1 diabetic patients (P < 0.01) and correlated with the levels of MCP-1 in this group (r = 0.726, P < 0.001). Arterial fibrosis correlated with IL-6 and MCP-1 levels (r = 0.411, P < 0.001 and r = 0.378, P = 0.001). A significant correlation was observed between VCAM-1 levels and both the degree of arterial narrowing and c-IMT. CONCLUSIONS: Type 1 diabetes produces a proinflammatory state in the arteries of end-stage CKD patients, with increased levels of IL-6, MCP-1, and VCAM-1, as well as a greater degree of p65 activation, which are associated with more severe vascular lesions and higher c-IMT. Although causality is not demonstrated, these findings support the major role of inflammation in type 1 diabetic patients with CKD.

Medial artery calcification of uremic patients: a histological, histochemical and ultrastructural study.

Recent findings suggest that vascular calcification (VC) is an active process similar to bone mineralization, the vascular smooth muscle cells (VSMCs) undergoing phenotypic differentiation into osteoblastic cells and synthesizing calcification-regulating proteins found in bone. This study has investigated the VC process of uremic patients, with a morphologic approach. Epigastric artery samples from 49 uremic, non-diabetic patients were taken during kidney transplantation. Sections from paraffin-embedded samples were stained with hematoxylin/eosin and von Kossa. CD68 was immunohistochemically detected, and sections from frozen samples were stained with Oil Red O. Deeply calcified samples were stained with Picrosirius Red, PAS, and Alcian blue. Specimens from one patient with moderate and one with severe VC were examined under the electron microscope. None of the samples had atherosclerosis. Calcifications were found in the media of 38 patients. In 23, dot-like calcifications were irregularly scattered near the adventitia (light VC); in 11, granular calcifications formed concentric rings near the adventitia (moderate-advanced VC); in 4, zones of consolidated calcifications were found (severe VC). These zones were poor in collagen, glycoproteins and proteoglycans. In cases with moderate or severe VC, VSCMs showed necrotic changes. Matrix vesicles could be recognized in the extracellular spaces. In cases with severe VC, uncalcified or partially calcified membranous bodies were found, together with Liesegang rings. Patches of fibrin were also found. These findings point to a mainly degenerative mechanism of VC, which proceeds from the outer portion of the media. An active mechanism, however, cannot be excluded. A unifying hypothesis is suggested.

Medial artery calcification in ESRD patients is associated with deposition of bone matrix proteins.

BACKGROUND: In non-ESRD patients, recent studies have demonstrated that the process of vascular calcification resembles developmental osteogenesis. Patients with ESRD are known to have excessive vascular calcification, but this has previously been attributed to the non-cell-mediated process of metastatic calcification. METHODS: To determine if the calcification observed in patients with ESRD is related to a cell-mediated process, we removed a piece of inferior epigastric artery at the time of renal transplant. Calcium content of the entire vessel was quantified with spiral computed tomography (CT). The vessel was then examined histologically for calcification and the presence of bone matrix proteins by immunohistochemistry, and medial and intimal thickness quantified by histomorphometry. These findings were correlated with demographic, clinical and laboratory values. RESULTS: The proximal inferior epigastric artery was obtained from 41 patients undergoing renal transplantation, but two were inadequate for histologic examination. Twenty-seven of the remaining vessels had no evidence of calcification by MacNeal's or Alizarin red pH 4.2 staining, five vessels had mild/moderate calcification, and seven had severe calcification, all in the medial layer. Calcification assessed histologically was closely correlated with calcification score as assessed by spiral CT, normalized for vessel weight (P=0.027). Positive immunostaining for the bone matrix proteins osteopontin, type I collagen, bone sialoprotein, and alkaline phosphatase was strongly correlated with calcification (all P < or = 0.001), as was a history of coronary artery disease (P < 0.001), and diabetes (P=0.034). The calcification score by spiral CT correlated with these same factors and the serum phosphorus and calcium x phosphorus product (P=0.032 and 0.037). The location of immunostaining for the bone proteins was strongly associated with the presence of calcification. However, positive immunostaining also was observed in association with disorganization of the vascular smooth muscle cells in the medial layer due to deposition of a matrix-like substance, prior to overt calcification. CONCLUSIONS: In patients with ESRD undergoing renal transplantation, vascular calcification of the medial layer of the inferior epigastric artery is common (44%), can be detected by spiral CT, and is associated with deposition of bone matrix proteins. This implies an active cell-mediated process, raising hope that directed intervention can arrest this process.