Reduced circulating CD63+ extracellular vesicle levels associate with atherosclerosis in hypercholesterolaemic mice and humans.

AIMS: The association and co-isolation of low-density lipoproteins (LDL) and extracellular vesicles (EVs) have been shown in blood plasma. Here we explore this relationship to better understand the role of EVs in atherogenesis. METHODS AND RESULTS: Wild type (WT), PCSK9-/-, and LDLR-/- C57BL/6 mice were used in this study. Eleven week-old male mice were fed high-fat diet (HFD) for 12 weeks or kept on normal diet until old age (22-months). Cardiac function was assessed by ultrasound, cholesterol was quantified with a colorimetric kit and circulating EVs were measured using flow cytometry. Plaques were analysed post-mortem using Oil-Red-O staining of the aortic arch. EVs were measured from platelet free blood plasma samples of normal and hypercholesterolaemic clinical patients. Based on annexin V and CD63 staining, we found a significant increase in EV levels in LDLR-/- and PCSK9-/- mice after HFD, but CD81 showed no significant change in either group. There was no significant change in plaque formation after HFD. PCSK9-/- mice show a favourable cardiac function after HFD. Blood cholesterol levels progressively increased during HFD, with LDLR-/- mice showing high levels while PCSK9-/- were significantly lowered compared to WT animals. In mice at old age, similar cholesterol levels were observed as in young mice. In old age, LDLR-/- mice showed significantly increased plaques. At old age, ejection fraction was decreased in all groups of mice, as were CD63+ EVs. Similarly to mice, in patients with hypercholesterolaemia, CD63+ EVs were significantly depleted. CONCLUSIONS: This research demonstrates an inverse relationship between circulating EVs and cholesterol, making EVs a potential marker for cardiovascular disease (CVD). HFD causes reduced cardiac function, but atherosclerotic development is slowly progressing in hypercholesterolaemic models and only observed with old animals. These results also bring further evidence for the benefit of using of PCSK9 inhibitors as therapeutic agents in CVD.

Laparoscopic approach for xanthogranulomatous pyelonephritis and pyonephrosis.

INTRODUCTION: Xanthogranulomatous pyelonephritis (XGPN) is a rare form of chronic renal inflammation, caused by long-term obstruction of the urinary tract. Pyonephrosis is a severe suppurative complication of acute obstructive pyelonephritis. Although minimally invasive approaches have many advantages, the safe dissection of the kidney may not be always achievable. MATERIALS AND METHODS: We reviewed 27 cases diagnosed with either XGPN or pyonephrosis, who underwent laparoscopic total nephrectomy between October 2016 and March 2022 in our department. All interventions were performed using the Karl Storz 3D laparoscopic system. The surgical approach was standard transperitoneal nephrectomy for the majority of XGPN, while pyonephrosis cases were carried out in a retroperitoneally. All procedures were performed or supervised by the same surgeon. RESULTS: The mean operative time was 269.85 minutes (range 145-360). The mean hemoglobin drop after surgery was 1.41 g/dl (range 0.3-2.3 g/dl). Difficult dissection was encountered in 13 cases (48.14%). Nine out of 13 interventions were carried out in a complete intracorporeal fashion, while conversion to open surgery was needed in 4 cases. Vascular complications involving the major blood vessels comprised of one case of inferior vena cava (IVC) tear. Digestive tract-related complications comprised two fistulas of the descending colon and one peritoneal breach. Multiorgan resection was performed in 6 cases. CONCLUSION: Total nephrectomy in cases of XGPN and pyonephrosis is a challenging procedure. The laparoscopic approach is feasible, as most complications are resolved intracorporeally. However, it may remain reserved for large-volume centers with experienced surgeons.

Faricimab Treat-and-Extend for Diabetic Macular Edema: Two-Year Results from the Randomized Phase 3 YOSEMITE and RHINE Trials.

PURPOSE: To evaluate the 2-year efficacy, durability, and safety of dual angiopoietin-2 and vascular endothelial growth factor (VEGF) A pathway inhibition with intravitreal faricimab according to a personalized treat-and-extend (T&E)-based regimen with up to every-16-week dosing in the YOSEMITE and RHINE (ClinicalTrials.gov identifiers, NCT03622580 and NCT03622593, respectively) phase 3 trials of diabetic macular edema (DME). DESIGN: Randomized, double-masked, noninferiority phase 3 trials. PARTICIPANTS: Adults with visual acuity loss (best-corrected visual acuity [BCVA] of 25-73 letters) due to center-involving DME. METHODS: Patients were randomized 1:1:1 to faricimab 6.0 mg every 8 weeks, faricimab 6.0 mg T&E (previously referred to as personalized treatment interval), or aflibercept 2.0 mg every 8 weeks. The T&E up to every-16-week dosing regimen was based on central subfield thickness (CST) and BCVA change. MAIN OUTCOME MEASURES: Included changes from baseline in BCVA and CST, number of injections, durability, absence of fluid, and safety through week 100. RESULTS: In YOSEMITE and RHINE (n = 940 and 951, respectively), noninferior year 1 visual acuity gains were maintained through year 2; mean BCVA change from baseline at 2 years (weeks 92, 96, and 100 average) with faricimab every 8 weeks (YOSEMITE and RHINE, +10.7 letters and +10.9 letters, respectively) or T&E (+10.7 letters and +10.1 letters, respectively) were comparable with aflibercept every 8 weeks (+11.4 letters and +9.4 letters, respectively). The median number of study drug injections was lower with faricimab T&E (YOSEMITE and RHINE, 10 and 11 injections, respectively) versus faricimab every 8 weeks (15 injections) and aflibercept every 8 weeks (14 injections) across both trials during the entire study. In the faricimab T&E arms, durability was improved further during year 2, with > 60% of patients receiving every-16-week dosing and approximately 80% receiving every-12-week or longer dosing at week 96. Almost 80% of patients who achieved every-16-week dosing at week 52 maintained every-16-week dosing without an interval reduction through week 96. Mean CST reductions were greater (YOSEMITE/RHINE weeks 92/96/100 average: faricimab every 8 weeks -216.0/-202.6 µm, faricimab T&E -204.5/-197.1 µm, aflibercept every 8 weeks -196.3/-185.6 µm), and more patients achieved absence of DME (CST < 325 µm; YOSEMITE/RHINE weeks 92-100: faricimab every 8 weeks 87%-92%/88%-93%, faricimab T&E 78%-86%/85%-88%, aflibercept every 8 weeks 77%-81%/80%-84%) and absence of intraretinal fluid (YOSEMITE/RHINE weeks 92-100: faricimab every 8 weeks 59%-63%/56%-62%, faricimab T&E 43%-48%/45%-52%, aflibercept every 8 weeks 33%-38%/39%-45%) with faricimab every 8 weeks or T&E versus aflibercept every 8 weeks through year 2. Overall, faricimab was well tolerated, with a safety profile comparable with that of aflibercept. CONCLUSIONS: Clinically meaningful visual acuity gains from baseline, anatomic improvements, and extended durability with intravitreal faricimab up to every 16 weeks were maintained through year 2. Faricimab given as a personalized T&E-based dosing regimen supports the role of dual angiopoietin-2 and VEGF-A inhibition to promote vascular stability and to provide durable efficacy for patients with DME. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

How to manage renal masses in kidney transplant recipients? A collaborative review by the EAU-YAU kidney transplantation and renal cancer working groups.

INTRODUCTION: Kidney transplant (KT) recipients have a four-times higher risk of renal malignancies compared to general population. As these patients frequently harbor bilateral or multifocal tumors, the management of renal masses is still under debate. OBJECTIVE: To explore the current management of the native kidney masses in KT patients. ACQUISITION OF EVIDENCE: We performed a literature search on MEDLINE/PubMed database. A number of 34 studies were included in the present review. SYNTHESIS OF EVIDENCE: In frail patients with renal masses below 3 cm, active surveillance is a feasible alternative. Nephron-sparing surgery is not justified for masses in the native kidney. Radical nephrectomy is the standard treatment for post-transplant renal tumors of the native kidneys in KT recipients, with laparoscopic techniques leading to significantly less perioperative complication rates as compared to the open approach. Concurrent bilateral native nephrectomy at the time of transplantation can be considered in patients with renal mass and polycystic kidney disease, especially if no residual urinary output is present. Patients with localized disease and successful radical nephrectomy do not require immunosuppression adjustment. In metastatic cases, mTOR agents can ensure efficient antitumoral response, while maintaining proper immunosuppression in order to protect the graft. CONCLUSIONS: Post-transplant renal cancer of the native kidneys is a frequent occurrence. Radical nephrectomy is most frequently performed for localized renal masses. A standardized and widely-approved screening strategy for malignancies of native renal units is yet to be implemented.

Formation of a protein corona on the surface of extracellular vesicles in blood plasma.

In this study we tested whether a protein corona is formed around extracellular vesicles (EVs) in blood plasma. We isolated medium-sized nascent EVs of THP1 cells as well as of Optiprep-purified platelets, and incubated them in EV-depleted blood plasma from healthy subjects and from patients with rheumatoid arthritis. EVs were subjected to differential centrifugation, size exclusion chromatography, or density gradient ultracentrifugation followed by mass spectrometry. Plasma protein-coated EVs had a higher density compared to the nascent ones and carried numerous newly associated proteins. Interactions between plasma proteins and EVs were confirmed by confocal microscopy, capillary Western immunoassay, immune electron microscopy and flow cytometry. We identified nine shared EV corona proteins (ApoA1, ApoB, ApoC3, ApoE, complement factors 3 and 4B, fibrinogen α-chain, immunoglobulin heavy constant γ2 and γ4 chains), which appear to be common corona proteins among EVs, viruses and artificial nanoparticles in blood plasma. An unexpected finding of this study was the high overlap of the composition of the protein corona with blood plasma protein aggregates. This is explained by our finding that besides a diffuse, patchy protein corona, large protein aggregates also associate with the surface of EVs. However, while EVs with an external plasma protein cargo induced an increased expression of TNF-α, IL-6, CD83, CD86 and HLA-DR of human monocyte-derived dendritic cells, EV-free protein aggregates had no effect. In conclusion, our data may shed new light on the origin of the commonly reported plasma protein 'contamination' of EV preparations and may add a new perspective to EV research.

Matrix metalloproteinase (MMP)-7 in Barrett's esophagus and esophageal adenocarcinoma: expression, metabolism, and functional significance.

Matrix metalloproteinase (MMP)-7, unlike many MMPs, is typically expressed in epithelial cells. It has been linked to epithelial responses to infection, injury, and tissue remodeling including the progression of a number of cancers. We have now examined how MMP-7 expression changes in the progression to esophageal adenocarcinoma (EAC), and have studied mechanisms regulating its expression and its functional significance. Immunohistochemistry revealed that MMP-7 was weakly expressed in normal squamous epithelium adjacent to EAC but was abundant in epithelial cells in both preneoplastic lesions of Barrett's esophagus and EAC particularly at the invasive front. In the stroma, putative myofibroblasts expressing MMP-7 were abundant at the invasive front but were scarce or absent in adjacent tissue. Western blot and ELISA revealed high constitutive secretion of proMMP-7 in an EAC cell line (OE33) that was inhibited by the phosphatidylinositol (PI) 3-kinase inhibitor LY294002 but not by inhibitors of protein kinase C, or MAP kinase activation. There was detectable proMMP-7 in cultured esophageal myofibroblasts but it was undetectable in media. Possible metabolism of MMP-7 by myofibroblasts studied by proteomic analysis indicated degradation via extensive endopeptidase, followed by amino- and carboxpeptidase, cleavages. Myofibroblasts exhibited increased migration and invasion in response to conditioned media from OE33 cells that was reduced by MMP-7 knockdown and immunoneutralization. Thus, MMP-7 expression increases at the invasive front in EAC which may be partly attributable to activation of PI 3-kinase. Secreted MMP-7 may modify the tumor microenvironment by stimulating stromal cell migration and invasion.

Increased prevalence of gallstone disease and impaired gallbladder motility in patients with Barrett's esophagus.

The prevalence of gallstones in patients with Barrett's esophagus (BE) and their gallbladder motility relative to that of healthy volunteers and GERD patients without BE were investigated. Of the 707 patients reviewed, 203 (125 males and 78 females) had BE. The prevalence of gallstones was significantly higher in the patients with BE than in those without BE (34 vs. 20%, respectively). The gallbladder functions of 22 patients with GERD, 27 patients with BE and 21 healthy volunteers were assessed by ultrasonography before and after a test meal. The patients with BE had significantly higher fasting volume and residual volume, but lower ejection volume, ejection fraction and ejection rate values than those of the healthy controls. None of the ultrasonographic parameters of patients without BE were significantly different from those of the controls. Patients with BE have a more complex gastrointestinal motility disorder that involves the gallbladder, and this makes this subset of patients with GERD more prone to gallstone disease.

Effect of calcium dobesilate on progression of early diabetic retinopathy: a randomised double-blind study.

BACKGROUND: The study was carried out to confirm the effect of calcium dobesilate (CaD) compared to placebo (PLA) on the blood-retinal barrier (BRB) permeability in early diabetic retinopathy (DR). METHODS: Adults with type II diabetes and early diabetic retinopathy (below level 47 of ETDRS grading and PVPR between 20 and 50x10(-6)/ min, plasma-free fluorescein) were included in this double-blind placebo-controlled study. Treatment was 2 g daily for 24 months. The primary parameter, posterior vitreous penetration ratio (PVPR), was measured every 6 months by fluorophotometry. Secondary parameters were fundus photography, fluorescein angiography and safety assessments. Metabolic control was performed every 3 months. RESULTS: A total of 194 patients started the treatment (98 CaD, 96 PLA) and 137 completed the 24-month study (69 CaD, 68 PLA). Both treatment groups were comparable at baseline, with ETDRS level 10 in about 59% of patients. Mean PVPR change from baseline after 24 months was significantly (P=0.002) lower in the CaD group [-3.87 (SD 12.03)] than in the PLA group [+2.03 (SD 12.86)], corresponding to a 13.2% decrease in the CaD group and a 7.3% increase in the PLA group. PVPR evolution was also analysed by HbA1c classes (<7%, between 7 and 9%, > or =9%) and results confirmed the superiority of CaD independently of the diabetes control level. A highly significant difference [CaD: -3.38 (SD 13.44) versus PLA: +3.50 (SD 13.70)] was also obtained in a subgroup of patients without anti-hypertensive and/or lipid-lowering agents (P=0.002 at 24 months). A further analysis of the secondary parameters showed significant changes in favour of CaD in the evolution from baseline to the last visit of haemorrhages (P=0.029), DR level (P=0.0006) and microaneurysms (P=0.013). Regarding safety, only 2.5% (n=5 patients/ events) of all adverse events reported were assessed as possibly or probably related to the test drug, while all serious adverse events were reported as unlikely. There was no statistical difference between groups. CONCLUSION: Calcium dobesilate 2 g daily for 2 years shows a significantly better activity than placebo on prevention of BRB disruption, independently of diabetes control. Tolerance was very good.

Expression of glutamate receptors on cultured cerebral endothelial cells.

Activation of glutamate receptors has been shown to mediate a large number of neuronal processes such as long-term potentiation and ischemic damage. In addition to neurons and glia, glutamate receptors may occur on cerebral endothelial cells (CECs). The aim of the present study was to determine which glutamate receptors are expressed in CECs and to demonstrate the functional presence of such channels. By using reverse transcriptase-polymerase chain reaction, we showed that primary cultures of rat CECs express N-methyl-D-aspartate (NMDA) receptors (NR1 subunit, which is necessary for the formation of functional NMDA receptors, and NR2A-C subunits), 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl-propionate (AMPA) receptors (GLUR1-4 subunits), and metabotropic receptors (mGLUR). Exposure of the cultures to 2 mM glutamate, a well-established mediator of ischemic damage, for 30 min increased significantly the phosphorylation of calcium/calmodulin-dependent protein kinase II even after 10- and 60-min recovery times. This effect could be prevented by the NMDA blocker MK-801. The presence of multiple glutamate receptor types may confer a finely tuned responsiveness of the cerebral endothelium to glutamate in physiological and pathological conditions.

Effect of subtype-specific Ca(2+)-antagonists and Ca(2+)-free media on the field stimulation-evoked release of ATP and [3H]acetylcholine from rat habenula slices.

The involvement of different subtypes of voltage-sensitive (Ca2+ channels in the initiation of field stimulation-induced endogenous adenosine triphosphate (ATP) and [3H]acetylcholine ([3H]ACh) release was investigated in the superfused rat habenula slices. ATP, measured by the luciferin-luciferase assay, and [3H]ACH were released simultaneously from the tissue in response to low frequency electrical stimulation (2 Hz, 2.5 msec, 360 shocks). The N-type Ca(2+)-channel blocker omega-conotoxin GVIA (omega-CgTX, 0.01-1 microM) reduced the stimulation-evoked release of ATP and [3H]ACh in a dose-dependent manner. Similarly, the P-type Ca2+ channel antagonist omega-agatoxin IVA (omega-Aga IVA) (0.05 microM) and the inorganic Ca(2+)-channel blocker Ca2+ (0.2 mM) inhibited the outflow of both transmitters, while Ni2+ (0.1 mM) was without significant effect. A high correlation was observed between the percent inhibition of ATP release and percent inhibition of ACh release caused by the different Ca2+ antagonists. Long-term perfusion (i.e., 90 min) with Ca(2+)-free solution inhibited the evoked-release of ATP and [3H]ACh. In contrast, perfusion of slices with the same media for a shorter time (i.e., 20 min) did not reduce the release of [3H]ACh and ATP but even increased the evoked-release of ATP about fourfold. The breakdown of extracellular ATP was not blocked under low [Ca2+]0 condition, measured by the creatine phosphokinase assay and HPLC-UV technique. Application of extra- or intracellular Ca2+ chelators, and dipyridamole (2 microM), the nucleoside transporter inhibitor, did not reduce the excess release of ATP after short-term perfusion with Ca(2+)-free media. Tetrodotoxin (TTX, 1 microM), while inhibiting the majority of ATP release under normal conditions, was also unable to reduce release under low [Ca2+]0 conditions. In summary, we showed that both N- and P-type Ca2+ channels are involved in the initiation of electrical stimulation-evoked release of ATP and [3H]ACh in the rat habenula under normal extracellular calcium concentration. Under low [CA2+]0 conditions an additional release of ATP occurs, which is not associated with action potential propagation.