Microplastics and Nanoplastics in Atheromas and Cardiovascular Events.

BACKGROUND: Microplastics and nanoplastics (MNPs) are emerging as a potential risk factor for cardiovascular disease in preclinical studies. Direct evidence that this risk extends to humans is lacking. METHODS: We conducted a prospective, multicenter, observational study involving patients who were undergoing carotid endarterectomy for asymptomatic carotid artery disease. The excised carotid plaque specimens were analyzed for the presence of MNPs with the use of pyrolysis-gas chromatography-mass spectrometry, stable isotope analysis, and electron microscopy. Inflammatory biomarkers were assessed with enzyme-linked immunosorbent assay and immunohistochemical assay. The primary end point was a composite of myocardial infarction, stroke, or death from any cause among patients who had evidence of MNPs in plaque as compared with patients with plaque that showed no evidence of MNPs. RESULTS: A total of 304 patients were enrolled in the study, and 257 completed a mean (±SD) follow-up of 33.7±6.9 months. Polyethylene was detected in carotid artery plaque of 150 patients (58.4%), with a mean level of 21.7±24.5 µg per milligram of plaque; 31 patients (12.1%) also had measurable amounts of polyvinyl chloride, with a mean level of 5.2±2.4 µg per milligram of plaque. Electron microscopy revealed visible, jagged-edged foreign particles among plaque macrophages and scattered in the external debris. Radiographic examination showed that some of these particles included chlorine. Patients in whom MNPs were detected within the atheroma were at higher risk for a primary end-point event than those in whom these substances were not detected (hazard ratio, 4.53; 95% confidence interval, 2.00 to 10.27; P<0.001). CONCLUSIONS: In this study, patients with carotid artery plaque in which MNPs were detected had a higher risk of a composite of myocardial infarction, stroke, or death from any cause at 34 months of follow-up than those in whom MNPs were not detected. (Funded by Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale and others; ClinicalTrials.gov number, NCT05900947.).

Glyoxalase 1 knockdown induces age-related ß-cell dysfunction and glucose intolerance in mice.

Tight control of glycemia is a major treatment goal for type 2 diabetes mellitus (T2DM). Clinical studies indicated that factors other than poor glycemic control may be important in fostering T2DM progression. Increased levels of methylglyoxal (MGO) associate with complications development, but its role in the early steps of T2DM pathogenesis has not been defined. Here, we show that MGO accumulation induces an age-dependent impairment of glucose tolerance and glucose-stimulated insulin secretion in mice knockdown for glyoxalase 1 (Glo1KD). This metabolic alteration associates with the presence of insular inflammatory infiltration (F4/80-positive staining), the islet expression of senescence markers, and higher levels of cytokines (MCP-1 and TNF-α), part of the senescence-activated secretory profile, in the pancreas from 10-month-old Glo1KD mice, compared with their WT littermates. In vitro exposure of INS832/13 ß-cells to MGO confirms its casual role on ß-cell dysfunction, which can be reverted by senolytic treatment. These data indicate that MGO is capable to induce early phenotypes typical of T2D progression, paving the way for novel prevention approaches to T2DM.

The urotensin-II receptor: A marker for staging and steroid outcome prediction in ulcerative colitis.

BACKGROUND: Urotensin-II receptor- (UTR) related pathway exerts a key-role in promoting inflammation. The aim was to assess the relationship between UTR expression and clinical, endoscopic and biochemical severity of ulcerative colitis (UC), exploring its predictivity of intravenous (iv) steroid administration therapeutic outcome. METHODS: One-hundred patients with first diagnosis of UC and 44 healthy subjects were enrolled. UTR expression was assessed by qPCR, Western Blot (WB) and immunohistochemistry (IHC). Clinical, endoscopic and histological activity of UC were evaluated by using Truelove and Witts (T&W) severity index, Mayo Endoscopic Score (MES), and Truelove and Richards Index (TRI). The partial and full Mayo scores (PMS and FMS) were assessed to stage the disease. RESULTS: The UTR expression, resulted higher in the lesioned mucosa of UC patients in comparison to healthy subjects (p < .0001 all). Direct relationship between UTR (mRNA and protein) expression and disease severity assessment (T&W, PMS, MES and TRI) was highlighted (p < .0001 all). UTR expression resulted also higher in the 72 patients requiring iv steroids administration compared to those who underwent alternative medications, (p < .0001). The 32 steroid-non-responders showed an increased UTR expression (WB, IHC and qPCR from lesioned mucosa), compared to 40 steroid-responders (p: .0002, .0001, p < .0001 respectively). The predictive role of UTR expression (p < .05) on the negative iv steroids administration therapeutic outcome was highlighted and ROC curves identified the thresholds expressing the better predictive performance. CONCLUSIONS: UTR represents a promising inflammatory marker related to clinical, endoscopic, and histological disease activity as well as a predictive marker of steroid administration therapeutic outcome in the UC context.

Sodium-glucose cotransporter-2 (SGLT2) expression in diabetic and non-diabetic failing human cardiomyocytes.

This study aimed at investigating the SGLT2 expression in human cardiomyocytes. Human studies evaluating cardiomyocyte SGLT2s expression are limited. To better clarify this issue, SGLT2 protein expression was assessed in human hearts of diabetic and non-diabetic patients, and in AC16 human cardiomyocyte cell line. A prospective study with a follow-up of patients who underwent their first heart transplant (HTX) was performed. Explanted heart, basal (1 week after HTX), and final (48 weeks after HTX) endomyocardial biopsies (EMBs) from patients were evaluated for SGLT2 occurrence in cardiomyocyte with immunohistochemistry, immunofluorescence and SGLT2 quantization with both real-time reverse transcription-polymerase chain reaction and Western blot analysis. The immunofluorescence co-localization of SGLT2 in cardiomyocyte evidenced that an increased expression in the explanted heart from diabetic patients compared to non-diabetic (p < 0.001). In all final EMBs from diabetic patients, the expression of SGLT2 in cardiomyocyte was increased compared to non-diabetic (p < 0.01). This evidence was confirmed by Western blot analysis of SGLT2 protein. In addition, PCR analysis revealed very low mRNA levels in basal EMBs from diabetic and non-diabetic patients (p = NS), whereas final EMBs from diabetic patients showed higher SGLT2 mRNA levels in diabetic compared to non-diabetic patients (p < 0.05). Cultured human cardiomyocytes exposed to high-glucose showed increased expression of SGLT2 protein compared to cells exposed to normal glucose (p < 0.05). The presence of SGLT2 in cardiomyocytes supports the hypothesis of SGLT2i-mediated impact on metabolic pathways within cardiomyocytes. Moreover, metabolic disorders linked to diabetes may lead promptly to upregulation of SGLT2 levels in human cardiomyocytes.

Systemic Beta-Hydroxybutyrate Affects BDNF and Autophagy into the Retina of Diabetic Mice.

Background: Diabetic retinopathy (DR) is a neurovascular disease, characterized by a deficiency of brain-derived neurotrophic factor (BDNF), a regulator of autophagy. Beta-hydroxybutyrate (BHB), previously reported as a protective agent in DR, has been associated with BDNF promotion. Here, we investigated whether systemic BHB affects the retinal levels of BDNF and local autophagy in diabetic mice with retinopathy; Methods: C57BL/6J mice were administered with intraperitoneal (i.p.) streptozotocin (STZ) (75 mg/kg) injection to develop diabetes. After 2 weeks, they received i.p. injections of BHB (25−50−100 mg/kg) twice a week for 10 weeks. Retinal samples were collected in order to perform immunofluorescence, Western blotting, and ELISA analysis; Results: BHB 50 mg/kg and 100 mg/kg significantly improved retinal BDNF levels (p < 0.01) in diabetic mice. This improvement was negatively associated with autophagosome−lysosome formations (marked by LC3B and ATG14) and to higher levels of connexin 43 (p < 0.01), a marker of cell integrity. Moreover, BHB administration significantly reduced M1 microglial activation and autophagy (p < 0.01); Conclusions: The systemic administration of BHB in mice with DR improves the retinal levels of BDNF, with the consequent reduction of the abnormal microglial autophagy. This leads to retinal cell safety through connexin 43 restoration.

N-palmitoyl-D-glucosamine, a Natural Monosaccharide-Based Glycolipid, Inhibits TLR4 and Prevents LPS-Induced Inflammation and Neuropathic Pain in Mice.

Toll-like receptors (TLRs) are key receptors through which infectious and non-infectious challenges act with consequent activation of the inflammatory cascade that plays a critical function in various acute and chronic diseases, behaving as amplification and chronicization factors of the inflammatory response. Previous studies have shown that synthetic analogues of lipid A based on glucosamine with few chains of unsaturated and saturated fatty acids, bind MD-2 and inhibit TLR4 receptors. These synthetic compounds showed antagonistic activity against TLR4 activation in vitro by LPS, but little or no activity in vivo. This study aimed to show the potential use of N-palmitoyl-D-glucosamine (PGA), a bacterial molecule with structural similarity to the lipid A component of LPS, which could be useful for preventing LPS-induced tissue damage or even peripheral neuropathies. Molecular docking and molecular dynamics simulations showed that PGA stably binds MD-2 with a MD-2/(PGA)3 stoichiometry. Treatment with PGA resulted in the following effects: (i) it prevented the NF-kB activation in LPS stimulated RAW264.7 cells; (ii) it decreased LPS-induced keratitis and corneal pro-inflammatory cytokines, whilst increasing anti-inflammatory cytokines; (iii) it normalized LPS-induced miR-20a-5p and miR-106a-5p upregulation and increased miR-27a-3p levels in the inflamed corneas; (iv) it decreased allodynia in peripheral neuropathy induced by oxaliplatin or formalin, but not following spared nerve injury of the sciatic nerve (SNI); (v) it prevented the formalin- or oxaliplatin-induced myelino-axonal degeneration of sciatic nerve. SIGNIFICANCE STATEMENT We report that PGA acts as a TLR4 antagonist and this may be the basis of its potent anti-inflammatory activity. Being unique because of its potency and stability, as compared to other similar congeners, PGA can represent a tool for the optimization of new TLR4 modulating drugs directed against the cytokine storm and the chronization of inflammation.

MicroRNA-33 and SIRT1 influence the coronary thrombus burden in hyperglycemic STEMI patients.

Primary percutaneous coronary intervention (PPCI) is a pivotal treatment in ST-segment elevation myocardial infarction (STEMI) patients. However, in hyperglycemic-STEMI patients, the incidence of death is still significant. Here, the involvement of sirtuin 1 (SIRT1) and miR33 on the pro-inflammatory/pro-coagulable state of the coronary thrombus was investigated. Moreover, 1-year outcomes in hyperglycemic STEMI in patients subjected to thrombus aspiration before PPCI were evaluated. Results showed that hyperglycemic thrombi displayed higher size and increased miR33, reactive oxygen species, and pro-inflammatory/pro-coagulable markers. Conversely, the hyperglycemic thrombi showed a lower endothelial SIRT1 expression. Moreover, in vitro experiments on endothelial cells showed a causal effect of SIRT1 modulation on the pro-inflammatory/pro-coagulative state via hyperglycemia-induced miR33 expression. Finally, SIRT1 expression negatively correlated with STEMI outcomes. These observations demonstrate the involvement of the miR33/SIRT1 pathway in the increased pro-inflammatory and pro-coagulable state of coronary thrombi in hyperglycemic STEMI patients.

Telmisartan cardioprotects from the ischaemic/hypoxic damage through a miR-1-dependent pathway.

The aim of this study was to investigate whether telmisartan protects the heart from the ischaemia/reperfusion damage through a local microRNA-1 modulation. Studies on the myocardial ischaemia/reperfusion injury in vivo and on the cardiomyocyte hypoxia/reoxygenation damage in vitro were done. In vivo, male Sprague-Dawley rats administered for 3 weeks with telmisartan 12 mg/kg/d by gastric gavage underwent ischaemia/reperfusion of the left descending coronary artery. In these rats, infarct size measurement, ELISA, immunohistochemistry (IHC) and reverse transcriptase real-time polymerase chain reaction showed that expressions of connexin 43, potassium voltage-gated channel subfamily Q member 1 and the protein Bcl-2 were significantly increased by telmisartan in the reperfused myocardium, paralleled by microRNA-1 down-regulation. In vitro, the transfection of cardiomyocytes with microRNA-1 reduced the expressions of connexin 43, potassium voltage-gated channel subfamily Q member 1 and Bcl-2 in the cells. Telmisartan (50 µmol/L) 60 minutes before hypoxia/reoxygenation, while not affecting the levels of miR-1 in transfected cells in normoxic condition, almost abolished the increment of miR-1 induced by the hypoxia/reoxygenation to transfected cells. All together, telmisartan cardioprotected against the myocardial damage through the microRNA-1 modulation, and consequent modifications of its downstream target connexin 43, potassium voltage-gated channel subfamily Q member 1 and Bcl-2.

Extrapleural solitary fibrous tumor: A distinct entity from pleural solitary fibrous tumor. An update on clinical, molecular and diagnostic features.

Solitary fibrous tumor (SFT) is a mesenchymal neoplasm that was originally described to be localized in the pleura, but thereafter, this has been reported in several anatomic sites. Although the etiology of the neoplasm remains largely unknown, the pathogenesis seems to be related to an NAB2-STAT6 fusion gene due to paracentric inversion on chromosome 12q13. The diagnosis of extrapleural SFT is challenging, owing to its rarity, and requires an integrated approach that includes specific clinical, histological, immunohistochemical, and even molecular findings. Histologically, extrapleural SFT shares morphological features same as those of the pleural SFT because it is characterized by a patternless distribution of both oval- and spindle-shaped cells in a variable collagen stroma. In addition, morphological variants of mixoid, fat-forming, and giant cell-rich tumors are described. A correct diagnosis is mandatory for a proper therapy and management of the patients with extrapleural SFT, as extrapleural SFT is usually more aggressive than pleural form, particularly cases occurring in the mediastinum, retroperitoneum, pelvis, and meninges. Although SFT is usually considered as a clinically indolent neoplasm, the prognosis is substantially unpredictable and only partially related to morphological features. In this context, cellularity, neoplastic borders, cellular atypias, and mitotic activity can show a wide range of variability. We review extrapleural SFT by discussing diagnostic clues, differential diagnosis, recent molecular findings, and prognostic factors.

Identification of perineural invasion at imaging staging as a novel potential risk factor in rectal cancer: A case report.

Treatment of rectal cancer has improved over the years thanks to a multidisciplinary approach. A correct staging has a fundamental role for risk stratification and to define the best treatment for each patient. Unfortunately, approximately 30% of patients with locally advanced rectal cancers will experience tumor recurrence. Thus, the identification of novel clinical-pathological and radiological prognostic factors represents an urgent unmet clinical need. Here we report the case of a patient with radically resected localized rectal cancer who developed an impressive early pelvic recurrence. To better understand the clinical scenario, we have studied the possible factors related to the aggressiveness of the disease. The only poor prognosticfactor that was evidenced at histological report was perineural invasion. Therefore, we questioned whether we could evaluate perineural invasion with imaging, similar to head and neck tumors. Learning from this clinical case, we believe that improving the risk stratification and radiology reporting is necessary to provide the best care for the patient and allow for a better prognosis prediction. Of course, our data should be considered as hypothesis generating and should be further investigated and validated in larger and prospective studies.

Melanocortin 3,5 receptors immunohistochemical expression in colonic mucosa of inflammatory bowel disease patients: A matter of disease activity?

BACKGROUND: Melanocortin 3 and 5 receptors (i.e., MC3R and MC5R) belong to the melanocortin family. However, data regarding their role in inflammatory bowel diseases (IBD) are currently unavailable. AIM: This study aims to ascertain their expression profiles in the colonic mucosa of Crohn's disease (CD) and ulcerative colitis (UC), aligning them with IBD disease endoscopic and histologic activity. METHODS: Colonic mucosal biopsies from CD/UC patients were sampled, and immunohistochemical analyses were conducted to evaluate the expression of MC3R and MC5R. Colonic sampling was performed on both traits with endoscopic scores (Mayo endoscopic score and CD endoscopic index of severity) consistent with inflamed mucosa and not consistent with disease activity (i.e., normal appearing mucosa). RESULTS: In both CD and UC inflamed mucosa, MC3R (CD: + 7.7 fold vs normal mucosa, P < 0.01; UC: + 12 fold vs normal mucosa, P < 0.01) and MC5R (CD: + 5.5 fold vs normal mucosa, P < 0.01; UC: + 8.1 fold vs normal mucosa, P < 0.01) were significantly more expressed compared to normal mucosa. CONCLUSION: MC3R and MC5R are expressed in the colon of IBD patients. Furthermore, expression may differ according to disease endoscopic activity, with a higher degree of expression in the traits affected by disease activity in both CD and UC, suggesting a potential use of these receptors in IBD pharmacology.

Presence of tumor deposits is a strong indicator of poor outcome in patients with stage III colorectal cancers undergoing radical surgery.

BACKGROUND: Tumor deposits (TDs) are emerging as an adverse prognostic factor in colorectal cancers (CRCs). However, TDs are somewhat neglected in the current staging system. It has been proposed either to add the TD count to the number of metastatic lymph nodes or to consider TDs as distant metastases; however, the scientific basis for these proposals seems questionable. This study aimed to investigate a new staging system. METHODS: A total of 243 consecutive patients with stage III CRC who were undergoing curative resection and adjuvant chemotherapy were included. Each substage of stage III TNM was split according to the absence or presence of TDs. Receiver operating characteristic (ROC) curves and bootstrap methods were used to compare the current vs the new competing staging system in terms of oncologic outcome prediction. RESULTS: A high rate of TDs was recorded (124 cases [51%]). TDs were correlated with other adverse prognostic indicators, particularly vascular and perineural invasions, and showed a negative correlation with the number of removed lymph nodes, suggesting a possible multimodal origin. In addition, TDs were confirmed to have a negative impact on oncologic outcome, regardless of their counts. Compared with the current staging system, the new classification displayed higher values at survival ROC analysis, a significantly better stratification of patients, and effective identification of patients at high risk of recurrence. CONCLUSIONS: TDs negatively affect the prognosis in CRCs. A revision of the staging system could be useful to optimize treatments. The proposed new classification is easy to implement and more accurate than the current one. This study was registered online on the ClinicalTrials.gov website under the following identifier: NCT05923450.

Ocular pharmacological and biochemical profiles of 6-thioguanine: a drug repurposing study.

Introduction: The purine analog 6-thioguanine (6TG), an old drug approved in the 60s to treat acute myeloid leukemia (AML), was tested in the diabetic retinopathy (DR) experimental in vivo setting along with a molecular modeling approach. Methods: A computational analysis was performed to investigate the interaction of 6TG with MC1R and MC5R. This was confirmed in human umbilical vein endothelial cells (HUVECs) exposed to high glucose (25 mM) for 24 h. Cell viability in HUVECs exposed to high glucose and treated with 6TG (0.05-0.5-5 µM) was performed. To assess tube formation, HUVECs were treated for 24 h with 6TG 5 µM and AGRP (0.5-1-5 µM) or PG20N (0.5-1-5-10 µM), which are MC1R and MC5R antagonists, respectively. For the in vivo DR setting, diabetes was induced in C57BL/6J mice through a single streptozotocin (STZ) injection. After 2, 6, and 10 weeks, diabetic and control mice received 6TG intravitreally (0.5-1-2.5 mg/kg) alone or in combination with AGRP or PG20N. Fluorescein angiography (FA) was performed after 4 and 14 weeks after the onset of diabetes. After 14 weeks, mice were euthanized, and immunohistochemical analysis was performed to assess retinal levels of CD34, a marker of endothelial progenitor cell formation during neo-angiogenesis. Results: The computational analysis evidenced a more stable binding of 6TG binding at MC5R than MC1R. This was confirmed by the tube formation assay in HUVECs exposed to high glucose. Indeed, the anti-angiogenic activity of 6TG was eradicated by a higher dose of the MC5R antagonist PG20N (10 µM) compared to the MC1R antagonist AGRP (5 µM). The retinal anti-angiogenic effect of 6TG was evident also in diabetic mice, showing a reduction in retinal vascular alterations by FA analysis. This effect was not observed in diabetic mice receiving 6TG in combination with AGRP or PG20N. Accordingly, retinal CD34 staining was reduced in diabetic mice treated with 6TG. Conversely, it was not decreased in diabetic mice receiving 6TG combined with AGRP or PG20N. Conclusion: 6TG evidenced a marked anti-angiogenic activity in HUVECs exposed to high glucose and in mice with DR. This seems to be mediated by MC1R and MC5R retinal receptors.

Assessment of the DNA Mismatch Repair System Is Crucial in Colorectal Cancers Necessitating Adjuvant Treatment: A Propensity Score-Matched and Win Ratio Analysis.

A deficient DNA mismatch repair (MMR) system is identified in a non-negligible part of sporadic colorectal cancers (CRCs), and its prognostic value remains controversial. High tumor mutational burden, along with a poor response to conventional chemotherapy and excellent results from immunotherapy, are the main features of this subset. The aim of this study was to evaluate the predictive value of DNA MMR system status for its best treatment. Four hundred and three CRC patients, operated on from 2014 to 2021 and not treated with immunotherapy, entered this study. Immunohistochemistry and polymerase chain reaction, as appropriate, were used to unequivocally group specimens into microsatellite stable (MSS) and instable (MSI) tumors. The win-ratio approach was utilized to compare composite outcomes. MSI tumors accounted for 12.9% of all series. The right tumor location represented the most important factor related to MSI. The status of the DNA MMR system did not appear to correlate with outcome in early-stage CRCs not requiring adjuvant treatment; in advanced stages undergoing conventional chemotherapy, MSI tumors showed significantly poorer overall and disease-free survival rates and the highest win ratio instead. The determination of DNA MMR status is crucial to recommending correct management. There is clear evidence that instable CRCs needing adjuvant therapy should undergo appropriate treatments.

Case report of unusual synchronous anal and rectal squamous cell carcinoma: clinical and therapeutic lesson.

Synchronous tumors of the rectum and anus are sporadic. Most cases in the literature are rectal adenocarcinomas with concomitant anal squamous cell carcinoma. To date, only two cases of concomitant squamous cell carcinomas of the rectum and anus are reported, and both were treated with up-front surgery and received abdominoperineal resection with colostomy. Here, we report the first case in the literature of a patient with synchronous HPV-positive squamous cell carcinoma of the rectum and anus treated with definitive chemoradiotherapy with curative intent. The clinical-radiological evaluation demonstrated complete tumor regression. After 2 years of follow-up, no evidence of recurrence was observed.

Evidence of an anti-inflammatory effect of PCSK9 inhibitors within the human atherosclerotic plaque.

BACKGROUND AND AIMS: Preclinical evidence suggests that proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors hold anti-inflammatory properties independently of their ability to lower LDL-cholesterol (C). However, whether PCSK9 inhibitors exert anti-inflammatory effects within the atherosclerotic plaque in humans is unknown. We explored the impact of PCSK9 inhibitors, used as monotherapy, compared with other lipid-lowering drugs (oLLD) on the expression of inflammatory markers within the plaque, assessing also the subsequent incidence of cardiovascular events. METHODS: In an observational study, we recruited 645 patients on stable therapy for at least six months and undergoing carotid endarterectomy, categorizing patients according to the use of PCSK9 inhibitors only (n = 159) or oLLD (n = 486). We evaluated the expression of NLRP3, caspase-1, IL-1ß, TNFα, NF-kB, PCSK9, SIRT3, CD68, MMP-9, and collagen within the plaques in the two groups through immunohistochemistry, ELISA, or immunoblot. A composite outcome including non-fatal myocardial infarction, non-fatal stroke, and all-cause mortality was assessed during a 678 ± 120 days follow-up after the procedure. RESULTS: Patients treated with PCSK9 inhibitors had a lower expression of pro-inflammatory proteins and a higher abundance of SIRT3 and collagen within the plaque, a result obtained despite comparable levels of circulating hs-CRP and observed also in LDL-C-matched subgroups with LDL-C levels <100 mg/dL. Patients treated with PCSK9 inhibitors showed a decreased risk of developing the outcome compared with patients on oLLD, also after adjustment for multiple variables including LDL-C (adjusted hazard ratio 0.262; 95% CI 0.131-0.524; p < 0.001). The expression of PCSK9 correlated positively with that of pro-inflammatory proteins, which burden was associated with a higher risk of developing the outcome, independently of the therapeutic regimen. CONCLUSIONS: The use of PCSK9 inhibitors is accompanied by a beneficial remodelling of the inflammatory burden within the human atheroma, an effect possibly or partly independent of their LDL-C lowering ability. This phenomenon might provide an additional cardiovascular benefit.