Toll-like receptors in experimental atherosclerosis

Aim: To study the effect of the activation of TLR2, TLR3, TLR4, TLR8 expression during the developmental process of atherosclerosis. Method: 24 male New Zealand White rabbits, with the body weight 2.5-3 Kg approximately were divided in 4 groups. Animals of the control group (E) received regular food for 3 months. Animals of groups A, B and C received high fat cholesterol diet (2%) for 1, 2 and 3 months, respectively. At the end of each experimental period animals were euthanized and a segment of the thoracic and abdominal aorta was removed and fixed in paraffin and in OCT. Histological sections were submitted to morphometric analysis (aorta thickness, % luminal radius, %content of collagen, calcium and elastin, % surface area). Segments of thoracic and abdominal aorta were, also, placed in RNA/DNA free eppendorfs for the mRNA analysis of the TLR2, TLR3, TLR4 and TLR8. Blood was withdrawn from the ocular artery of the animals before and at the end of each experimental period for the determination of cholesterol, triglycerides and homocysteine levels in the serum. Statistical evaluation of all data was performed with the use of the appropriate statistical packages. Results: A significant increase of the body weight in groups B and C was observed. Cholesterol levels were significantly higher in groups A,B,C (p<0,05) . Triglycerides levels were also elevated in animals of group C (p<0,05) and the homocysteine levels in groups B, C (p<0,05). The size of atherosclerotic lesions of the thoracic and abdominal aorta was increased in animals of groups B and C (p<0,05). The % of luminal radius of the thoracic aorta increased in animals of group C (p<0,05). Extensive calcification of atherosclerotic lesions was observed in the thoracic and abdominal aorta in animals of group C (p<0,05). The genome of the TLR8 in the rabbit model was designed. A significant increase of the mRNA levels of the TLR2, TLR8 was observed in segments of the thoracic aorta in animals of groups B and C (p<0,05). An increase of TLR3 levels was noticed in segments of thoracic aorta in groups A and B, while a similar increase of TLR4 levels was noticed in group B. An increase of the TLR2, TLR3, TLR4 and TLR8 was observed in the abdominal aorta in animals of group A (p<0,05) and TLR3 of those of group B (p<0,05). A non significant decrease of TLR2, 3, 4, 8 levels was noticed in segments of the abdominal aorta in animals of group C. Conclusion: It is concluded that Toll-like receptors are expressed during the process of atherosclerosis. There was a significant correlation between the type and the lesion of the atherosclerotic plaque and the levels of TLRs. The expression of the TLR8 was totally correlated with the extent of atherosclerotic lesion. It is postulated, that the decrease of mRNA levels of TLR3,4 in the thoracic aorta and of the TLR2,3,4,8 in the abdominal aorta is related with the response of the immune system. The increase of the TLR2, 8 in the thoracic aorta is, molecularly, correlated with the development of atherosclerosis and, morphometrically, with the extent of the atherosclerotic plaque.Η αθηρωμάτωση αποτελεί, στις αναπτυγμένες χώρες, την κύρια αιτία των καρδιαγγειακών παθήσεων. Η κατανόηση, κατά την τελευταία δεκαετία, των μοριακών και κυτταρικών μηχανισμών πρόκλησης της αθηρωματικής βλάβης έχει μεταβληθεί ριζικά λόγω της σημαντικής προόδου της μοριακής βιολογίας και της γενετικής καθώς και της επιτυχούς ανάπτυξης ζωικών προτύπων αθηρωμάτωσης. Η αθηρωμάτωση δεν αποτελεί, πλέον, μια αναπόφευκτη συνέπεια της γήρανσης αλλά μια χρόνια φλεγμονώδη ασθένεια με εξαιρετικά υψηλή νοσηρότητα και θνητότητα. Τα τελευταία χρόνια, η έρευνα στην διευκρίνιση του μηχανισμού της αθηρογένεσης εστιάζεται και στους υποδοχείς Τoll-like receptors (TLR) και στον τρόπο με τον οποίο αυτοί μπορεί να ενεργοποιήσουν, να ενισχύσουν ή να παρατείνουν την φλεγμονώδη αντίδραση στο τοίχωμα των αρτηριών. Παρά το γεγονός ότι πολλές έρευνες αποδεικνύουν την ενεργοποίηση των TLRs στα κυτταρικά συστατικά του καρδιαγγειακού συστήματος φαίνεται ότι μεμονωμένα οικογενειακά μέλη των TLR διαδραματίζουν ένα παθοφυσιολογικό ρόλο στην πρόκληση των καρδιαγγειακών παθήσεων και για τον λόγο αυτό επιδιώκεται να καθοριστούν κλινικά. Η μελέτη σε βασικό αλλά και σε κλινικό επίπεδο για την οικογένεια αυτή των TLRs έχουν υποβοηθήσει στην κατανόηση του μηχανισμού της αγγειακής και της μυοκαρδιακής λειτουργίας. Ο σκοπός της παρούσας μελέτης ήταν να διευκρινιστεί αν η παρατεταμένη ενεργοποίησή του TLR8 καθώς και των TLR2, TLR3, TLR4 συμβάλει στην ανάπτυξη ή την επιδείνωσή της αθηρωματικής βλάβης. Ο στόχος της ερευνητικής προσπάθειας ήταν: α) να σχεδιαστεί ο TLR8 στο γονιδίωμα του κουνελιού, β) να προσδιοριστεί ο ρόλος των TLR υποδοχέων στην ανάπτυξη της αθηρωματικής νόσου στο υπερλιπιδαιμικό κουνέλι, γ) να μελετηθεί η έκφραση των υποδοχέων TLR στο αρτηριακό τοίχωμα κατά την εξελικτική πορεία της αθηρωμάτωσης και δ) να συσχετιστούν οι TLR με το είδος και την έκταση των αθηρωματικών αλλοιώσεων. Πρόκειται για μία προοπτική, τυχαιοποιημένη και ελεγχόμενη μελέτη παρέμβασης στην οποία χρησιμοποιήθηκαν ως ζωικά πρότυπα 24 αρσενικά κουνέλια Νέας Ζηλανδίας. Την 12η εβδομάδα ηλικίας τους τα κουνέλια τυχαιοποιήθηκαν στις παρακάτω ομάδες: α) στην ομάδα (Ε) ελέγχου (n=6), β) στην ομάδα Α (n=6), γ) στην ομάδα Β (n=6) και δ) στην ομάδα Γ (n=6). Στις ομάδες Α, Β και Γ χορηγήθηκε αθηρογόνος τροφή και ειδικότερα στην ομάδα Α για 4 εβδομάδες, στην ομάδα Β για 8 εβδομάδες και στην ομάδα Γ για 12 εβδομάδες. Στην ομάδα Ε (n=6) χορηγήθηκε φυσιολογική τροφή για διάστημα 12 εβδομάδων. Στο τέλος της χρονικής περιόδου κάθε ομάδας έγινε ευθανασία των ζωικών προτύπων. Τμήμα της θωρακικής και της κοιλιακής αορτής αφαιρέθηκε και εγκλείστηκε σε παραφίνη ενώ ένα άλλο τμήμα φυλασσόταν σε ειδικά φιαλίδια στους -80°C για τον προσδιορισμό των TLR υποδοχέων. Τόσο κατά την έναρξη όσο και κατά την λήξη της μελέτης έγινε αιμοληψία στα ζώα κάθε ομάδας για τον προσδιορισμό των επιπέδων της χοληστερίνης, των τριγλυκεριδίων και της ομοκυστεΐνης. Κατά την ιστολογική ανάλυση προσδιορίστηκαν η διάμετρος του αυλού του αγγείου το εμβαδό των αθηρωματικών πλακών και ο % βαθμός στένωσης του αυλού που προκαλείται από τις αθηρωματικές πλάκες. Με την χρώση Alizarin Red μελετήθηκε η ασβεστοποίηση των αθηρωματικών πλακών, με την χρώση Οil red η συσσώρευση των λιπιδίων στην αθηρωματική πλάκα και τέλος με την χρώση ορσεΐνης το εμβαδό της ίνωσης. ................................................................

Myocardial Expression of Pluripotency, Longevity, and Proinflammatory Genes in the Context of Hypercholesterolemia and Statin Treatment

Background: This study sought to assess the effect of statin therapy on myocardial inflammation in a White New Zealand rabbit model of atherogenesis. Methods: The mRNA expression levels of pro-inflammatory, pluripotency, and aging-related markers were quantified following a controlled feeding protocol and statin treatments. Results: Following high-cholesterol diet induction, we observed significant upregulation in the myocardial mRNA levels of MYD88, NF-κB, chemokines (CCL4, CCL20, and CCR2), IFN-γ, interleukins (IL-1β, IL-2, IL-4, IL-8, IL-10, and IL-18), and novel markers (klotho, KFL4, NANOG, and HIF1α). In contrast, HOXA5 expression was diminished following a hyperlipidemic diet. Both statin treatments significantly influenced the markers studied. Nevertheless, rosuvastatin administration resulted in a greater reduction in MYD88, NF-kB, chemokines (CCL4, CCL20, and CCR2), and interleukins IL-1β, IL-8, KLF4, NANOG, and HIF1α than fluvastatin. Fluvastatin, on the other hand, led to a stronger decrease in IL-4. Downregulation of IL-2 and IL-18 and upregulation of IFNβ and HOXA5 were comparable between the two statins. Notably, rosuvastatin had a stronger effect on the upregulation of klotho and IL-10. Conclusion: Overall, statin therapy significantly attenuated inflammatory, pluripotency, and klotho expression in myocardial tissue under atherogenic conditions. Our findings also highlight the differential efficacy of rosuvastatin over fluvastatin in curtailing proatherogenic inflammation, which could have profound implications for the clinical management of cardiovascular disease

Surgery for metachronous oligometastatic esophageal cancer: Is there enough evidence?

Esophageal cancer is the sixth most common cause of cancer-related mortality worldwide. Despite advances in diagnostic modalities and treatment options, five-year survival rates are below 20%. Esophagectomy with extended lymph node dissection is the mainstay of treatment. More than 50% of patients experience recurrence within 1-3 years postoperatively. Recurrent disease may present locoregionally at the site of anastomosis or as recurrence through lymphatic spread in lymph node basins, as hematogenic metastasis, or as a combination of these. The standard treatment of recurrence is currently predicated on systemic chemotherapy and/or radiotherapy. Recent evidence suggests that surgical treatment of metachronous oligometastatic disease may be prognostically advantageous over medical management alone. Given the considerably low response rates to chemoradiotherapy, many institutions have adopted surgical treatment strategies for oligo-recurrent disease on a case-by-case basis. The aim of this article is to review the current evidence on the role of surgical treatment for metachronous oligometastases from esophageal cancer

Surgery for metachronous oligometastatic esophageal cancer: Is there enough evidence?

Esophageal cancer is the sixth most common cause of cancer-related mortality worldwide. Despite advances in diagnostic modalities and treatment options, five-year survival rates are below 20%. Esophagectomy with extended lymph node dissection is the mainstay of treatment. More than 50% of patients experience recurrence within 1-3 years postoperatively. Recurrent disease may present locoregionally at the site of anastomosis or as recurrence through lymphatic spread in lymph node basins, as hematogenic metastasis, or as a combination of these. The standard treatment of recurrence is currently predicated on systemic chemotherapy and/or radiotherapy. Recent evidence suggests that surgical treatment of metachronous oligometastatic disease may be prognostically advantageous over medical management alone. Given the considerably low response rates to chemoradiotherapy, many institutions have adopted surgical treatment strategies for oligo-recurrent disease on a case-by-case basis. The aim of this article is to review the current evidence on the role of surgical treatment for metachronous oligometastases from esophageal cancer. (C) 2021 AEC. Published by Elsevier Espana, S.L.U. All rights reserved

The role of exercise training and the endocannabinoid system in atherosclerotic plaque burden and composition in Apo-E-deficient mice

Introduction: We investigated the effect of combining exercise training and treatment with an endocannabinoid receptor 1 inhibitor (Rimonabant) on atherosclerosis burden and composition. Methods: Forty-eight apolipoprotein E-deficient (ApoE-/-) mice were kept on a 16-week high-fat diet. Mice were then placed on a normal diet and were randomized to the following groups with n=12 mice for 6 more weeks: 1) Control (Co) - no intervention; 2) Exercise (Ex) - exercise training on treadmill; 3) Rimonabant (Ri) - oral administration of rimonabant (10 mg/kg/day); or 4) Rimonabant+Exercise (RiEx) - combination of Ri and Ex groups treatment. At the end, all animals were sacrificed, and blood samples, as well as aortic root specimens, were obtained for histomorphometric analysis and quantification of the serum and plaque content of matrix metalloproteinases (MMPs). Results: The mean plaque area was significantly smaller (RiEx: 43.18±1.72%, Ri: 44.66±3.1%, Ex: 49±4.10%, Co: 70.43±2.83%) in all active treatment groups relative to the Co group (p<0.01). Conversely, the relative concentrations of collagen and elastin were increased significantly across all treatment groups compared to Co (p<0.05). Immunohistochemical analysis revealed significantly reduced macrophage content within plaques after all interventions, with the most pronounced effect observed after combined treatment (RiEx: 9.4±3.92%, Ri: 15±2.45%, Ex: 19.78±2.79%, Co: 34.25±4.99%; p<0.05). Within plaques, the TIMP-1 concentration was significantly upregulated in exercise-treated groups. MMP-3 and MMP-9 concentrations were equivalently decreased in all three active treatment groups compared to controls (p<0.001). Discussion: Both exercise and rimonabant treatments induced plaque regression and promoted plaque stability. The combined treatment failed to show additive or synergistic benefits relative to either intervention alone

Therapeutic potential of a distinct population of human amniotic fluid mesenchymal stem cells and their secreted molecules in mice with acute hepatic failure

Background There is increasing interest in the therapeutic potential of human mesenchymal stem cells (hMSCs), especially in diseases such as acute hepatic failure (AHF) that are predominantly caused by a variety of drugs and viruses. In previous studies, a distinct population termed human spindle-shaped MSCs were isolated and expanded from second trimester amniotic fluid (AF-MSCs) and characterised based on their phenotype, pluripotency and differentiation potential. Methods AF-MSCs, hepatic progenitor-like (HPL) cells and hepatocyte-like (HL) cells derived from AF-MSCs were transplanted into CCl4-injured NOD/SCID mice with the AHF phenotype in order to evaluate their therapeutic potential. Conditioned medium (CM) derived from AF-MSCs or HPL cells was then delivered intrahepatically in order to determine whether the engraftment of the cells or their secreted molecules are the most important agents for liver repair. Results Both HPL cells and AF-MSCs were incorporated into CCl4-injured livers; HPL cell transplantation had a greater therapeutic effect. In contrast, HL cells failed to engraft and contribute to recovery. In addition, HPL-CM was found to be more efficient than CM derived from AF-MSCs in treatment of the liver. Proteome profile analysis of HPL-CM indicated the presence of anti-inflammatory factors such as interleukins IL-10, IL-1ra, IL-13 and IL-27 which may induce liver recovery. Blocking studies of IL-10 secretion from HPL cells confirmed the therapeutic significance of this cytokine in the AHF mouse model. Conclusions Human spindle-shaped AF-MSCs or HPL cells might be valuable tools to induce liver repair and support liver function by cell transplantation. More importantly, the factors they release may also play an important role in cell treatment in diseases of the liver

Mechanosensitive Stem-Cell Genes and Klotho in Atherosclerotic Aortas: Regulating Spatially Deranged Expression Patterns Using Colchicine Regimens

Aims: Inflammatory dysregulation of mechanosensitive developmental genes may be central to atherogenesis. In the present seven-week model, we utilized colchicine regimens to curtail aortic atherogenesis in New Zealand White rabbits. We also explored the effect of colchicine regimens on atheroprotective (Klotho, HOXA5, NOTCH1, and OCT4) and proatherogenic (HIF1a, SOX2, BMP4, and NANOG) genes. Methods: The control (n = 6) and group A (n = 6) received standard and cholesterol-enriched chow, respectively. Groups B (n = 8) and C (n = 8) were fed hypercholesterolemic diet and were treated with colchicine plus fenofibrate or N-acetylcysteine (NAC), respectively. Results: Group A developed significantly greater thoracic and abdominal aortic atherosclerosis compared to groups B (p &lt; 0.001) and C (p &lt; 0.001). Combining colchicine with NAC resulted in stronger atheroprotection both in the thoracic and the abdominal aorta. In group A thoracic aortas, Klotho was downregulated compared to controls (95% CI: 1.82&ndash;15.76). Both colchicine regimens upregulated Klotho back to baseline levels (p &lt; 0.001). Colchicine/fenofibrate also significantly upregulated thoracic NOTCH1 compared to controls (95% CI: &minus;8.09 to &minus;0.48). Colchicine/NAC significantly reduced thoracic NANOG expression compared to hyperlipidemic diet alone (95% CI: 0.37&ndash;8.29). In the abdominal aorta, hypercholesterolemic diet resulted in significant downregulation of HOXA5 (95% CI: 0.03&ndash;2.74) which was reversed with colchicine/NAC back to baseline (95% CI: &minus;1.19 to 1.51). Colchicine/fenofibrate downregulated HIF1a compared to baseline (95% CI: 0.83&ndash;6.44). No significant differences were noted in terms of BMP4, SOX2, and OCT4. Conclusions: Overall, the aortic expression pattern of mechanosensitive genes seems to be spatially influenced by a hyperlipidemic diet and can be modified using colchicine-based therapy

Feasibility and Short-Term Outcomes of Three-Dimensional Hand-Sewn Esophago-Jejunal Anastomosis in Completely Laparoscopic Total Gastrectomy for Cancer

Laparoscopic total gastrectomy is on the rise. One of the most technically demanding steps of the approach is the construction of esophago-jejunal anastomosis. Several laparoscopic anastomotic techniques have been described, like linear stapler side-to-side or circular stapler end-to-side anastomosis; limited data exist regarding hand-sewn esophago-jejunal anastomosis. The study took place between January 2018 and June 2021. Patients enrolled in this study were adults with proximal gastric or esophago-gastric junction Siewert type III tumors that underwent 3D-assisted laparoscopic total gastrectomy. A hand-sewn esophago-jejunal anastomosis was performed in all cases laparoscopically. Forty consecutive cases were performed during the study period. Median anastomotic suturing time was 55 min, with intra-operative methylene blue leak test being negative in all cases. Median operating time was 240 min, and there were no conversions to open. The anastomotic leak rate and postoperative stricture rate were zero. The 30- and 90-day mortality rates were zero. Laparoscopic manual esophago-jejunal anastomosis utilizing a 3D platform in total gastrectomy for cancer can be performed with excellent outcomes regarding anastomotic leak and stricture rate. This anastomotic approach, although technically challenging, is safe and reproducible, with prominent results that can be disseminated in the surgical community

Activated Clotting Time as a Marker of Inflammation in Hospitalized Patients

Inflammation and coagulation pathways are implicated in circulatory disease, but their interaction has not been completely deciphered yet. In this study, we investigated the association of coagulation and inflammation indices (activated clotting time [ACT], C-reactive protein, neutrophils) in hospitalized patients. Blood samples were drawn from consecutive patients at admission and at 48 hours for the assessment of the aforementioned parameters (n = 63). Healthy controls matched for sex and age were also examined (n = 39). Activated clotting time positively correlated with CRP on admission (r = 0.354, P = .005), while the correlation was more robust on the second day (r = 0.775, P &lt; .001). Activated clotting time was significantly more prolonged in patients with abnormal CRP or abnormal absolute neutrophil count compared to patients with normal inflammatory markers (U = 55.0, P &lt; .001 and U = 310.5, P = .035, respectively). At 48 hours, a positive relationship was observed between ACT and relative percentage of neutrophils (r = 0.358, P = .004). These findings suggest a link between ACT and inflammation indices for the first time in humans. Further research is needed to determine whether these interrelations can be used to improve patient management

Feasibility and Short-Term Outcomes of Three-Dimensional Hand-Sewn Esophago-Jejunal Anastomosis in Completely Laparoscopic Total Gastrectomy for Cancer

Simple Summary Laparoscopic total gastrectomy for the treatment of gastric and esophago-gastric junction cancer is on the rise. The procedure&apos;s rate-limiting step is the construction of the esophago-jejunal anastomosis. Most surgeons are performing the anastomosis laparoscopically by utilizing an endoscopic linear or circular stapler; scarce evidence exists regarding laparoscopic hand-sewn esophago-jejunal anastomosis. Herein, we present our technique and results of laparoscopic manual esophago-jejunal anastomosis during totally laparoscopic total gastrectomy for gastric and esophago-gastric junction cancer. Anastomosis was performed in a two-layer fashion. Overall implementation provided excellent surgical outcomes in our cohort of patients; median anastomotic time was 55 min, while median operating time was 240 min. Intra-operative methylene blue leak test was negative in all cases. No anastomotic leak or anastomotic stricture were noted postoperatively. The 30-day and 90-day mortality rates were zero. Laparoscopic total gastrectomy is on the rise. One of the most technically demanding steps of the approach is the construction of esophago-jejunal anastomosis. Several laparoscopic anastomotic techniques have been described, like linear stapler side-to-side or circular stapler end-to-side anastomosis; limited data exist regarding hand-sewn esophago-jejunal anastomosis. The study took place between January 2018 and June 2021. Patients enrolled in this study were adults with proximal gastric or esophago-gastric junction Siewert type III tumors that underwent 3D-assisted laparoscopic total gastrectomy. A hand-sewn esophago-jejunal anastomosis was performed in all cases laparoscopically. Forty consecutive cases were performed during the study period. Median anastomotic suturing time was 55 min, with intra-operative methylene blue leak test being negative in all cases. Median operating time was 240 min, and there were no conversions to open. The anastomotic leak rate and postoperative stricture rate were zero. The 30- and 90-day mortality rates were zero. Laparoscopic manual esophago-jejunal anastomosis utilizing a 3D platform in total gastrectomy for cancer can be performed with excellent outcomes regarding anastomotic leak and stricture rate. This anastomotic approach, although technically challenging, is safe and reproducible, with prominent results that can be disseminated in the surgical community