Overexpression of Toll-Like Receptors 2, 3, 4, and 8 Is Correlated to the Vascular Atherosclerotic Process in the Hyperlipidemic Rabbit Model: The Effect of Statin Treatment.

BACKGROUND: Atherosclerosis is the major cause of cardiovascular disease; hypercholesterolemia is a major risk factor. We hypothesized that specific TLR members (TLR2, TLR3, TLR4, TLR8) may play a role in atherosclerosis progression and its accompanying inflammatory response. We determined the association of atherosclerotic lesions and TLR mRNA expression in different aortic sites. We also assessed the effects of fluvastatin (Flu) treatment on TLR expression and plaque characteristics. METHODS: Male rabbits, fed with an atherogenic diet for a duration of 3 months, were screened for advanced atherosclerotic lesions in the aorta. Additional animals received normal diet or normal diet plus Flu for 1 additional month. TLR mRNA expression in various thoracic and abdominal aortic segments was assessed, together with atherosclerotic changes. RESULTS: After high lipid diet, the atherosclerotic burden increased more in the abdominal than in the thoracic aorta; TLR2, 3, 4, and 8 also increased significantly. Flu decreased atherosclerotic plaque, calcium deposition, lipid cores, intraplaque hemorrhage, erythrocyte membranes, endothelial cells, and macrophage infiltration, while increasing smooth muscle cells in plaques of both aortic segments; it also lowered TLR2, 3, 4, and 8 expression in all aortic segments to a stronger degree than resumption of normal diet. There was a strong association between blood and tissue parameters during experimental period and finally a strong correlation found between these parameters with mRNA of TLR2, 3, 4, and 8 in various stages. CONCLUSION: For the first time TLR2, 3, 4, and 8 mRNA expression is prospectively explored after hypercholesterolemic diet in the rabbit model. TLR2, 3, 4, and 8 mRNA expression is strongly upregulated and correlates with the progression of atherosclerosis in the aorta. Flu significantly inhibited this progress and reduced inflammation via TLR downregulation which was strongly associated with regression of plaque morphology and atherosclerosis promoting factors.

Remote Ischemic Preconditioning May Attenuate Renal Ischemia-Reperfusion Injury in a Porcine Model of Supraceliac Aortic Cross-Clamping.

AIM: The effect of remote ischemic preconditioning (RIPC) in decreasing renal ischemia-reperfusion injury (IRI) during a suprarenal aortic cross-clamping was examined in a swine model. MATERIALS AND METHODS: Four groups of pigs were examined: (a) ischemia-reperfusion (IR) group, renal IRI produced by 30 min of supraceliac aortic cross-clamping; (b) RIPC I group, the same renal IRI following RIPC by brief occlusion of the infrarenal aorta (15 min ischemia and 15 min reperfusion); (c) RIPC II group, the same renal IRI following RIPC by brief occlusion of the infrarenal aorta (3 cycles of 5 min ischemia and 5 min reperfusion); (d) sham group. Renal function was assessed before and after IRI by examining creatinine, neutrophil gelatinase-associated lipocalin (NGAL), TNF-α, malondialdehyde (MDA), cystatin C and C-reactive protein (CRP) from renal vein blood samples at specific time intervals. RESULTS: Both RIPC groups presented significantly less impaired results compared to the IR group when considering MDA, cystatin C, CRP and creatinine. Between the two RIPC groups, RIPC II presented a better response with regard to CRP, NGAL, TNF-α, MDA and cystatin C. CONCLUSIONS: Remote IR protocols and mainly repetitive short periods of cycles of IR ameliorate the biochemical kidney effects of IRI in a model of suprarenal aortic aneurysm repair.

UGT1A1*28 polymorphism in chronic lymphocytic leukemia: the first investigation of the polymorphism in disease susceptibility and its specific cytogenetic abnormalities.

Chronic lymphocytic leukemia (CLL) has been recently attributed to a combination of genetic predisposition and exposure to environmental factors. UDP-glucuronosyltransferase (UGT)1A1*28 is an inborn polymorphism that results in significant downregulation of uridine diphosphate glucuronyltransferase 1-1 (UGT1A1) activity, one of the most critical metabolizing enzymes involved in the detoxification of toxic substances, some of which contribute to CLL pathogenesis. Here, for the first time, we investigated the putative impact of UGT1A1*28 on CLL incidence and on the formation of the most common chromosomal abnormalities of CLL. UGT1A1*28 was investigated in 109 CLL patients and 108 healthy controls, and was associated with karyotypic and fluorescence in situ hybridization (FISH) results. A significant high frequency of the mutant genotype was observed in patients carrying abnormal FISH patterns, especially del(11q) and +12, which are CLL-specific abnormalities. We also observed a significant association between UGT1A1*28 and the intermediate to unfavorable cytogenetic CLL risk groups. No difference, though, was observed in genotypes between patients and controls. Therefore, we could suggest that UGT-deficient individuals may be at a greater risk for developing CLL-specific abnormalities. Our study might serve as a starting point to consider UGT1A1*28 polymorphism as one of the possible predisposing factors of CLL pathogenesis.

Vascular endothelial growth factor receptors 1,3 and caveolin-1 are implicated in colorectal cancer aggressiveness and prognosis--correlations with epidermal growth factor receptor, CD44v6, focal adhesion kinase, and c-Met.

Vascular endothelial growth factor receptor-1 (VEGFR-1) and caveolin-1 have been shown to act both as tumor-promoting and tumor-suppressing proteins in various malignancies as well as in colorectal cancer (CRC), while VEGFR-3's lymphagiogenic involvement and connection to tumor parameters has yielded heterogenic results. This study was designed to investigate the expression of these molecules in 183 human CRC tissue specimens and explore their effect in both clinicopathological parameters and disease prognosis. We also utilize our previous results regarding epidermal growth factor receptor (EGFR), c-Met, CD44v6, and focal adhesion kinase, in an attempt to further clarify their distinct role in tumor prognosis and their crosstalk. Caveolin-1 was more freely distributed in the neoplasms of the right colon and restricted towards the left and the rectal cancer samples (p = 0.022); VEGFR-3 was associated with higher nodal metastasis' status (p = 0.001) and staging (p = 0.006), and loss of VEGFR-1 predicted distant metastasis (p = 0.026) and advanced stage (p = 0.049). Prompted by previous reports, we performed all analyses also in the patient group of early (I and II) tumor stage where it was evident that VEGFR-1 was more frequently expressed in patients under 60 years old (p = 0.014) and VEGFR-3 was significantly elevated in left colon cancers (p = 0.039) and female patients (p = 0.038). Within the advanced stage (III and IV), the absence of VEGFR-1 exhibited a tendency for higher M status (p = 0.067) and lack of caveolin-1 signified worse AJCC classification (p = 0.053). Additionally, patient survival was influenced from VEGFR-3 (p = 0.019) for the whole sample, whereas subgroup analyses provided a correlation between caveolin-1 expression and improved survival in the early detection group of patients (p = 0.022). Using Cox regression for all available markers, FAK, CD44v6, and Caveolin-1 [corrected] emerged in this study as potential surrogate markers, the latter having positive prognostic significance. We further explored the multiple receptor correlations that were identified.

Impact of donor and recipient age difference on long-term allograft survival after living donor renal transplantation: analysis of 478 cases.

INTRODUCTION: Either deceased or living-related renal transplantation constitutes the best therapeutic option for patients with end-stage renal disease. In this retrospective study, an attempt to identify parameters that affect allograft survival in living donor renal transplantation was made. METHODS: Between January 2000 and July 2012, 478 adult patients received a renal transplant from a living-related donor in our center and their records were retrospectively reviewed in November 2012. Data concerning donor age, recipient age, donor/recipient age difference, donor/recipient gender, and ABO compatibility/incompatibility were recorded and associated with renal allograft survival rate. RESULTS: Renal allograft survival rate was 96%, 89.5%, and 77.7% in the first, fifth, and 10th yr after transplantation, respectively. Only the difference between donor and recipient age was statistically significant in relation to graft survival. In cases with age difference >13 yr, graft survival rate was lower from the third yr onward. CONCLUSIONS: Only the age difference between donor and recipient exerts an adverse impact on graft outcome after living donor renal transplantation, whereas donor age, recipient age, donor/recipient gender, and ABO incompatibility do not significantly influence renal allograft survival.

FAK, CD44v6, c-Met and EGFR in colorectal cancer parameters: tumour progression, metastasis, patient survival and receptor crosstalk.

PURPOSE: Research for reliable and patient-specific markers in colorectal cancer (CRC) is based on solid evidence that staging alone is not informative enough. Employing four cellular receptors, we embarked to identify aggressive tumour behaviour and impact of surrogate marker expression on patient prognosis. METHODS: One-hundred eighty-three CRC patients were enrolled in our investigation that focused on an array of biological markers, namely epidermal growth factor receptor (EGFR), c-Met, focal adhesion kinase (FAK) and CD44v6. Tissue samples, clinicopathological data and patient's follow-up information were collected, and immunohistochemical assays evaluated the levels of the aforementioned molecules. All available data were correlated with tumour grade, stage, patient age, gender and survival. RESULTS: Expression of all receptors correlated closely with tumour stage (P < 0.01) exhibiting a connection with cancer's invasiveness and progress. Survival also proved to depend significantly on molecular expression (log-rank test for Kaplan-Meier; EGFR P = 0.030, c-Met P = 0.050, FAK P < 0.001, CD44v6 P < 0.001). Stage, FAK and CD44v6 emerged as independent predictors of survival in a stepwise regression analysis (FAK P = 0.001 Exp(B) = 2.517, 95 % confidence interval (CI) = 1.704-5.831 and CD44v6 P = 0.005, Exp(B) = 2.299, 95 % CI = 1.287-4.110). T-stage, nodal metastasis, all metastatic types (N/M) and size correlated with at least one of the receptors or their co-expression. Notably, increased staining for each receptor was followed by statistically significant expression elevation of at least one of the other markers. CONCLUSIONS: Our results suggest that the selected cellular receptors are suitable for use as biomarkers of survival and tumour progression in CRC. Furthermore, we provide additional evidence for receptor interaction, properly clarifying their importance, which could potentially lead to more effective anti-CRC regimens.

Adipokines: a novel link between adiposity and carotid plaque vulnerability.

BACKGROUND: In patients with carotid stenosis, we prospectively investigated the association of novel adipokines, apelin and visfatin, with gray-scale median (GSM) score, a valid index of carotid plaque vulnerability. We also assessed the impact of atorvastatin therapy on the above biochemical and imaging markers. MATERIALS AND METHODS: Seventy-four overweight [body-mass index (BMI) > 25 kg/m(2) , fat-mass > 30%], statin-free patients, with carotid stenosis, but without indications for intervention were enrolled. Thirty-eight age-, sex- and BMI-matched healthy subjects served as healthy controls (HC). All patients received gradual titrated (10-80 mg) atorvastatin therapy to target LDL-C < 100 mg/dL. GSM score, blood pressure (BP), fat-mass, lipid profile, and serum high-sensitivity C-reactive protein (hsCRP), apelin and visfatin levels were obtained at baseline and after 24 months. RESULTS: At baseline, patients with carotid atherosclerosis had worse lipid profile, lower apelin and higher systolic BP, hsCRP, visfatin levels compared with HC (P < 0·05). Notably, decreased apelin (P < 0·001) and GSM score (P = 0·010), while increased visfatin (P = 0·019) and hsCRP (P = 0·039) levels were found in symptomatic rather than asymptomatic patients. At baseline, GSM score correlated with fat-mass, BMI, LDL-C, visfatin and apelin (P < 0·05). Apelin, visfatin and fat-mass remained independent determinants of baseline GSM score (R(2) = 0·391, P = 0·007). In parallel, we found that apelin increment and LDL-C reduction were independently associated with the atorvastatin-induced GSM increase (R(2) = 0·411, P = 0·011). CONCLUSION: Increased fat-mass, low apelin and high visfatin serum levels seem to correlate with carotid plaque vulnerability in patients with carotid stenosis. The atorvastatin-induced modification of apelin and LDL-C may beneficially affect carotid plaque stability.

Prognostic significance of transforming growth factor beta (TGF-ß) signaling axis molecules and E-cadherin in colorectal cancer.

The transforming growth factor beta (TGF-ß) signaling pathway has been considered both a tumor suppressor and a cancer promoter. Additionally, downregulation of cell adhesion molecules such as E-cadherin plays an important role in the metastatic potential of colorectal cancer (CRC). The aim of the present study was to evaluate TGF-ß, TGF-ß type I receptor (TGF-ßR1), TGF-ß type II receptor (TGF-ßR2), Smad4, pSmad2/3, and E-cadherin expression in colorectal carcinoma and to correlate the obtained data with other standard prognostic parameters, such as disease stage, metastases, and patient survival. TGF-ß, TGF-ßR1, TGF-ßR2, Smad4, pSmad2/3, and E-cadherin expression was evaluated immunohistochemically in 195 unrelated CRC specimens and the results subjected to various statistical analyses. TGF-ß was expressed in 71.28%, TGF-ßR1 in 61.0%, TGF-ßR2 in 54.4%, Smad4 in 61.5%, pSmad2/3 in 71.3%, and E-cadherin in 50.26% of the colorectal carcinoma samples tested. The correlation of immunoexpression with the clinicopathological parameters of CRC revealed that the high expression of TGF-ß and low expression of TGF-ßR1, TGF-ßR2, Smad4, pSmad2/3, and E-cadherin were correlated with tumor-node-metastasis (TNM) stage of disease. High TGF-ß expression and low TGF-ßR1, TGF-ßR2, Smad4, and pSmad2/3 expression were also correlated with lymph node metastasis. The Kaplan-Meier survival curves demonstrated a clear association of cancer-specific overall survival with high TGF-ß, as well as low TGF-ßR1, TGF-ßR2, Smad4, pSmad2/3, and E-cadherin expression. Our results suggest that TGF-ß, TGF-ßR1, TGF-ßR2, Smad4, pSmad2/3, and E-cadherin are closely related to TNM stage of CRC. Moreover, TGF-ß, TGF-ßR2, Smad4, pSmad2/3, and E-cadherin emerge as valuable independent biomarkers of prognosis in CRC patients.

The beneficial effects of a direct thrombin inhibitor, dabigatran etexilate, on the development and stability of atherosclerotic lesions in apolipoprotein E-deficient mice : dabigatran etexilate and atherosclerosis.

PURPOSE: Dabigatran etexilate (DE) constitutes a novel, direct thrombin inhibitor. Regarding the association of thrombin with atherogenesis, we assessed the effects of DE on the development and stability of atherosclerotic lesions in apolipoprotein-E deficient (ApoE-/-) mice. MATERIALS-METHODS: Fifty male ApoE-/- mice were randomized to receive western-type diet either supplemented with DE 7.5 mg DE/g chow) (DE-group, n = 25) or matching placebo as control (CO-group, n = 25) for 12 weeks. After this period, all mice underwent carotid artery injury with ferric chloride and the time to thrombotic total occlusion (TTO) was measured. Then, mice were euthanatized and each aortic arch was analyzed for the mean plaque area, the content of macrophages, elastin, collagen, nuclear factor kappaB (NFκB), vascular cell adhesion molecule-1 (VCAM-1), matrix metalloproteinase-9 (MMP-9) and its inhibitor (TIMP-1). RESULTS: DE-group showed significantly longer TTO compared to CO-group (8.9 ± 2.3 min vs 3.5 ± 1.1 min, p < 0.001) and the mean plaque area was smaller in DE-group than CO-group (441.00 ± 160.01 × 10(3) µm(2) vs 132.12 ± 32.17 × 10(3) µm(2), p < 0.001). Atherosclerotic lesions derived from DE-treated mice showed increased collagen (p = 0.043) and elastin (p = 0.031) content, thicker fibrous caps (p < 0.001) and reduced number of internal elastic lamina ruptures per mm of arterial girth (p < 0.001) when compared to CO-group. Notably, DE treatment seemed to promote plaque stability possibly by reducing concentrations of NFκB, VCAM-1, macrophages and MMP-9 and increasing TIMP-1 within atherosclerotic lesions (p < 0.05). CONCLUSIONS: DE attenuates arterial thrombosis, reduces lesion size and may promote plaque stability in ApoE-/- mice. The plaque-stabilizing effects of chronic thrombin inhibition might be the result of the favorable modification of inflammatory mechanisms.

Acute histological changes of the lung after experimental Fontan circulation in a swine model.

BACKGROUND: Histological changes of the lungs were studied after the establishment of a modified total cavopulmonary connection (TCPC) without the use of cardiopulmonary bypass (CPB) or other means of temporary bypass on a swine model. MATERIAL/METHODS: 8 open chest-anesthetized pigs Landrace x Large White pigs (mean weight 43kg, mean age 4.5 months) underwent TCPC by the use of an appropriate size Y-shaped conduit connecting the superior and inferior caval veins (end-to-end anastomosis) to the pulmonary trunk (end-to-side anastomosis). After sternotomy, a wedge resection of the lung parenchyma was performed at baseline. Hemodynamic stability was sustained after TCPC establishment and 2 hours later another wedge resection of the lung was performed (from the same anatomic area). Histological studies were conducted by hematoxylin and eosin staining. RESULTS: All samples (n=8) at baseline were consistent with normal lung parenchyma. After the establishment of TCPC, all samples (n=8) revealed moderate mononuclear infiltration adjacent to pulmonary alveoli and bronchioles, findings compatible with bronchiolitis. CONCLUSIONS: In a normal swine model, 2 hours after the establishment of Fontan circulation without the use of CPB, pathologic examination of the lungs revealed bronchiolitis. Further research is needed to clarify these findings and the potential implications to the Fontan circulation, either immediate or long-term.

The impact of aerobic exercise training on novel adipokines, apelin and ghrelin, in patients with type 2 diabetes.

BACKGROUND: Accumulating data support the atheroprotective role of the novel adipokines, apelin and ghrelin. The aim of the present randomized study was to investigate the effects of aerobic exercise training on these adipokines in patients with type 2 diabetes mellitus (T2DM). MATERIAL/METHODS: Fifty-four overweight (BMI >25 kg/m²) patients with T2DM, but without vascular complications, were randomized to either the aerobic exercise training group (EG, N=27), 4 times/week, 45-60 min/session; or to the control group (CG, N=27), orally instructed to increase physical activity. Clinical glycemic and lipid parameters, exercise capacity (VO2peak), insulin, HOMA-IR, and serum levels of apelin and ghrelin were assessed at baseline and after 12 weeks. RESULTS: Aerobic exercise significantly improved lipid and glycemic profile and insulin sensitivity compared to CG (p<0.05). Furthermore, between-groups comparison showed a considerable exercise-induced upregulation in apelin (p=0.007) and VO2peak (p<0.001) levels. Negligible changes in body-weight, waist-hip ratio and ghrelin concentrations were detected within and between groups after the completion of the study (p>0.05). However, subgroup analysis revealed a considerable increment in ghrelin levels only in the exercise-treated women compared to their control counterparts (p=0.038). LDL and HOMA-IR reduction were found to be independent predictors of apelin increment in multiple regression analysis (R²=0.391, p=0.011). CONCLUSIONS: In patients with T2DM, systemic, long-term, aerobic exercise exerts positive effects on apelin and ghrelin (only in women), even in the absence of significant weight loss, suggesting its pleiotropic effects.

Experimentally modified Fontan circulation in an adolescent pig model without the use of cardiopulmonary bypass.

BACKGROUND: The feasibility and the hemodynamic outcome of Fontan circulation, without the use of cardiopulmonary bypass, were studied on a beating heart of an adolescent pig model, using a modified total cavopulmonary connection. MATERIAL/METHODS: Eight open-chest anesthetized pigs underwent a successful total cavopulmonary connection with the use of an appropriate Y-shaped Dacron-type conduit. Through a median sternotomy, the distal part of the superior vena cava was anastomosed end-to-end to one side of the conduit. The other side of the graft was anastomosed end-to-side to the main pulmonary artery. The conduit was tailored to an appropriate length and anastomosed end-to-end to the inferior vena cava. The hemodynamic status of the animals was recorded before and after the establishment of the total cavopulmonary connection. RESULTS: Forty-five minutes after completion of total cavopulmonary connection, and for a total of 1 hour, hemodynamic measurements showed a decrease in mean arterial and mean pulmonary artery pressures, heart rate and cardiac output. The inferior vena caval pressure and total pulmonary vascular resistance were increased. CONCLUSIONS: A total cavopulmonary connection, performed on a beating heart, without extracorporeal circulation or other means of temporary bypass, although it is technically demanding, is feasible.

Gracilis myocutaneous flap: evaluation of potential risk factors and long-term donor-site morbidity.

This study reviewed our experience with the gracilis myocutaneous (GMC) flap, potential risk factors for flap necrosis, and long-term morbidity at the donor-site. From 1993 to 2002, 29 GMC flaps were harvested from 27 patients (pedicled n = 21 and free n = 8). The overall incidence of flap necrosis was 13.79% (partial (n = 2) and total (n = 2) necrosis). Flap necrosis was correlated with body mass index >25 (P = 0.022), with smoking (P = 0.04 9) and with radiation therapy at the recipient site (P = 0.020). The long-term morbidity at the donor-site was low, except for scar appearance (17.24%), thigh contour deformity (58.62%), and hypoesthesia (17.24%). Significant age and gender differences were seen for ranking of scar ugliness, with females (P = 0.0061) and younger patients (age ≤55) (P = 0.046) assigned higher values. Significant age differences were seen for ranking of thigh contour deformity, with younger patients assigned higher values (P = 0.0012). In conclusion, patient overweight, smoking, and previous radiation therapy at the recipient site may be the "potential risk factors" for flap necrosis. The long-term morbidity at the donor-site was low, which was in agreement with previous reported studies. A larger series would be the subject of a future study.

Morphologic alterations of the genital mesentery implicated in testis non-descent in rats prenatally exposed to flutamide.

BACKGROUND: There is an endless debate on whether androgens mediate testis descent through developmental changes in the gubernacular or the cranial suspensory ligament. OBJECTIVE: To investigate the relation of any possible morphologic changes in the genital mesentery, that is, the system of genital peritoneal folds including the gubernacular and cranial suspensory ligaments, with the event of testis non-descent in rats prenatally exposed to the antiandrogen flutamide. MATERIALS AND METHODS: Time-pregnant Sprague Dawley rats received flutamide (100 mg/kg/d) or vehicle subcutaneously on gestational days 16-17. Flutamide-treated male offspring (n = 67), and vehicle-treated male (n = 34) and female (n = 28) offspring were surgically explored under microscope on postnatal day 50. Testicular position was examined bilaterally. Dimensions of genital mesentery parts were also assessed bilaterally. Association of flutamide-induced morphologic changes with descended (n = 61) and undescended (n = 50; 33 cryptorchid and 17 ectopic) testes was investigated with logistic regression analysis. RESULTS: The male genital mesentery comprised a cranial and a caudal fold converging on the vas deferens. Flutamide resulted in enlarged cranial and reduced caudal folds. Of all flutamide-induced alterations, the increased length of the posterior fixation of the cranial fold and the decreased length of the gubernacular ligament of the caudal fold were found to independently increase the odds of testis non-descent. Testicular ectopy, unlike cryptorchidism, was associated with a short gubernacular ligament only. The female genital mesentery consisted of a cranial fold only. CONCLUSION: Our findings showed a combined contribution of both cranial and caudal folds of the genital mesentery to testis non-descent, through an abnormally long mesentery root and an abnormally short gubernacular ligament, respectively. Inhibition of male-specific development of the genital mesentery with flutamide did not result in a feminized architecture.

Improved Repopulation Efficacy of Decellularized Small Diameter Vascular Grafts Utilizing the Cord Blood Platelet Lysate.

BACKGROUND: The development of functional bioengineered small-diameter vascular grafts (SDVGs), represents a major challenge of tissue engineering. This study aimed to evaluate the repopulation efficacy of biological vessels, utilizing the cord blood platelet lysate (CBPL). METHODS: Human umbilical arteries (hUAs, n = 10) were submitted to decellularization. Then, an evaluation of decellularized hUAs, involving histological, biochemical and biomechanical analysis, was performed. Wharton's Jelly (WJ) Mesenchymal Stromal Cells (MSCs) were isolated and characterized for their properties. Then, WJ-MSCs (1.5 × 106 cells) were seeded on decellularized hUAs (n = 5) and cultivated with (Group A) or without the presence of the CBPL, (Group B) for 30 days. Histological analysis involving immunohistochemistry (against Ki67, for determination of cell proliferation) and indirect immunofluorescence (against activated MAP kinase, additional marker for cell growth and proliferation) was performed. RESULTS: The decellularized hUAs retained their initial vessel's properties, in terms of key-specific proteins, the biochemical and biomechanical characteristics were preserved. The evaluation of the repopulation process indicated a more uniform distribution of WJ-MSCs in group A compared to group B. The repopulated vascular grafts of group B were characterized by greater Ki67 and MAP kinase expression compared to group A. CONCLUSION: The results of this study indicated that the CBPL may improve the repopulation efficacy, thus bringing the biological SDVGs one step closer to clinical application.

Predictive significance of preoperative serum VEGF-C and VEGF-D, independently and combined with Ca19-9, for the presence of malignancy and lymph node metastasis in patients with gastric cancer.

BACKGROUND: Cumulative evidence demonstrate that lymphangiogenic vascular endothelial growth factors (VEGF)-C and -D are over-expressed and associated to lymph node metastasis (LNM) in gastric cancer. The aim of this study is to investigate whether preoperative serum levels of VEGF-C and VEGF-D could be useful tumor markers in patients with operable gastric adenocarcinoma. METHODS: We prospectively examined serum samples from 40 patients and 40 non-cancer controls using enzyme-linked immunosorbent assay (ELISA). Logistic regression analysis was implemented. VEGF-C and VEGF-D were studied independently and in combination with Ca19-9. RESULTS: In gastric cancer patients, preoperative VEGF-C was significantly lower as compared to controls and to postoperative VEGF-C (P < 0.001); preoperative VEGF-D was significantly higher as compared to controls and to postoperative VEGF-D (P < 0.001). ROC curve analysis identified a VEGF-C/VEGF-D cut-off value of < 2.7 for the presence of gastric cancer, with 83% sensitivity and 75% specificity (P < 0.001). Backward stepwise selection modeling including sex, age, VEGF-D and Ca19-9, predicted the presence of LNM with 86% sensitivity and 82% specificity (P < 0.001). CONCLUSION: Circulating levels of VEGF-C and VEGF-D could play a role as biomarkers for serological detection and staging in gastric cancer.

Future Perspectives in Small-Diameter Vascular Graft Engineering.

The increased demands of small-diameter vascular grafts (SDVGs) globally has forced the scientific society to explore alternative strategies utilizing the tissue engineering approaches. Cardiovascular disease (CVD) comprises one of the most lethal groups of non-communicable disorders worldwide. It has been estimated that in Europe, the healthcare cost for the administration of CVD is more than 169 billion €. Common manifestations involve the narrowing or occlusion of blood vessels. The replacement of damaged vessels with autologous grafts represents one of the applied therapeutic approaches in CVD. However, significant drawbacks are accompanying the above procedure; therefore, the exploration of alternative vessel sources must be performed. Engineered SDVGs can be produced through the utilization of non-degradable/degradable and naturally derived materials. Decellularized vessels represent also an alternative valuable source for the development of SDVGs. In this review, a great number of SDVG engineering approaches will be highlighted. Importantly, the state-of-the-art methodologies, which are currently employed, will be comprehensively presented. A discussion summarizing the key marks and the future perspectives of SDVG engineering will be included in this review. Taking into consideration the increased number of patients with CVD, SDVG engineering may assist significantly in cardiovascular reconstructive surgery and, therefore, the overall improvement of patients' life.

Advanced Therapy Medicinal Products Challenges and Perspectives in Regenerative Medicine.

Recently, the design and development of a modern health policy in the field of regenerative medicine leads to the formation of a new and integrated cognitive field, which requires systematic research and study in order to produce innovative answers and best practices. Advanced therapy medicinal products (ATMPs) is a new product category, which is at the heart of concern since it has to deal with diseases in which traditional medicine has proven to be ineffective so far. The aim of this review is to provide evidence for the state of the art ATMPs and their modern applications in the field of regenerative medicine. The ATMPs are characterized by a great heterogeneity and variation in methods of isolation, which cover the entire spectrum from a single intravenous injection to a surgical placement. Clinical development of ATMP encounters specific challenges due to the nature of the product and the limited availability of non-clinical data. The gold standard of a controlled, randomized, clinical trial may not be feasible or ethically justified for all indications, particularly in life-threatening diseases, where there is no satisfactory standard of care. Therefore, the European Commission (EC) took initiatives in order to set standards and operating rules concerning authorization and supervision of ATMPs and on pharmacovigilance in relation to them. The European Union (EU) Regulation 1394/2007 provides the possibility of exceptions. In particular, the "hospital exemption" allows for the administration of an ATMP without a license on certain conditions. Although the Regulation 1394/2007 has led to the commercial exploitation of ATMPs, the reality today, 11 years after its first implementation, is completely different. While the Committee for Advanced Therapies (CAT) has already registered 285 products as ATMPs, only 10 licenses were granted which only remained six (the rest related to products withdrawn). The key players in the development and delivery of ATMPs still remain the academic/research centers and small and medium-sized enterprises; while the involvement of pharmaceutical companies is focusing on recent developments in the treatment of oncological incidents with in vitro modified cytotoxic T lymphocytes, and chimeric antigen receptor (CAR)-T cells.

Efficient differentiation of vascular smooth muscle cells from Wharton's Jelly mesenchymal stromal cells using human platelet lysate: A potential cell source for small blood vessel engineering.

BACKGROUND: The development of fully functional small diameter vascular grafts requires both a properly defined vessel conduit and tissue-specific cellular populations. Mesenchymal stromal cells (MSCs) derived from the Wharton's Jelly (WJ) tissue can be used as a source for obtaining vascular smooth muscle cells (VSMCs), while the human umbilical arteries (hUAs) can serve as a scaffold for blood vessel engineering. AIM: To develop VSMCs from WJ-MSCs utilizing umbilical cord blood platelet lysate. METHODS: WJ-MSCs were isolated and expanded until passage (P) 4. WJ-MSCs were properly defined according to the criteria of the International Society for Cell and Gene Therapy. Then, these cells were differentiated into VSMCs with the use of platelet lysate from umbilical cord blood in combination with ascorbic acid, followed by evaluation at the gene and protein levels. Specifically, gene expression profile analysis of VSMCs for ACTA2, MYH11, TGLN, MYOCD, SOX9, NANOG homeobox, OCT4 and GAPDH, was performed. In addition, immunofluorescence against ACTA2 and MYH11 in combination with DAPI staining was also performed in VSMCs. HUAs were decellularized and served as scaffolds for possible repopulation by VSMCs. Histological and biochemical analyses were performed in repopulated hUAs. RESULTS: WJ-MSCs exhibited fibroblastic morphology, successfully differentiating into "osteocytes", "adipocytes" and "chondrocytes", and were characterized by positive expression (> 90%) of CD90, CD73 and CD105. In addition, WJ-MSCs were successfully differentiated into VSMCs with the proposed differentiation protocol. VSMCs successfully expressed ACTA2, MYH11, MYOCD, TGLN and SOX9. Immunofluorescence results indicated the expression of ACTA2 and MYH11 in VSMCs. In order to determine the functionality of VSMCs, hUAs were isolated and decellularized. Based on histological analysis, decellularized hUAs were free of any cellular or nuclear materials, while their extracellular matrix retained intact. Then, repopulation of decellularized hUAs with VSMCs was performed for 3 wk. Decellularized hUAs were repopulated efficiently by the VSMCs. Biochemical analysis revealed the increase of total hydroyproline and sGAG contents in repopulated hUAs with VSMCs. Specifically, total hydroxyproline and sGAG content after the 1st, 2nd and 3rd wk was 71 ± 10, 74 ± 9 and 86 ± 8 µg hydroxyproline/mg of dry tissue weight and 2 ± 1, 3 ± 1 and 3 ± 1 µg sGAG/mg of dry tissue weight, respectively. Statistically significant differences were observed between all study groups (P < 0.05). CONCLUSION: VSMCs were successfully obtained from WJ-MSCs with the proposed differentiation protocol. Furthermore, hUAs were efficiently repopulated by VSMCs. Differentiated VSMCs from WJ-MSCs could provide an alternative source of cells for vascular tissue engineering.

Vitrified Human Umbilical Arteries as Potential Grafts for Vascular Tissue Engineering.

BACKGROUND: The development of a biological based small diameter vascular graft (d < 6 mm), that can be properly stored over a long time period at - 196 °C, in order to directly be used to the patients, still remains a challenge. In this study the decellularized umbilical arteries (UAs) where vitrified, evaluated their composition and implanted to a porcine model, thus serving as vascular graft. METHODS: Human UAs were decellularized using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS) detergents. Then, vitrified with vitrification solution 55 (VS55) solution, remained for 6 months in liquid nitrogen and their extracellular matrix composition was compared to conventionally cryopreserved UAs. Additionally, total hydroxyproline, sulphated glycosaminoglycan and DNA content were quantified in all samples. Finally, the vitrified umbilical arteries implanted as common carotid artery interposition graft to a porcine animal model. RESULTS: Decellularized and vitrified UAs characterized by proper preservation of extracellular matrix proteins and tissue architecture, whereas conventionally cryopreserved samples exhibited a disorganized structure. Total hydroxyproline content was preserved, although sulphated glycosaminoglycan and DNA contents presented significantly alterations in all samples. Implanted UAs successfully recellularized and remodeled as indicated by the histological analysis. CONCLUSION: Decellularized and vitrified UAs retained their structure function properties and can be possible used as an alternative source for readily accessible small diameter vascular grafts.