Heterogeneity and Differentiation of the Human Arterial Tree: Focus on microRNA Expression in Vascular Disease.

Human arteries show structural and functional peculiarities according to the nutrient and oxygen needs of a specific vascular district. This architectural heterogeneity is reflected in the pathological setting of cardiovascular diseases (CVDs). Indeed, the responsiveness to cardiovascular risk factors, and the morphological and molecular patterns are discriminating factors among CVDs affecting different vascular beds. MicroRNAs (miRNAs) are endogenous regulators of gene expression and fine-tuners of vascular cell differentiation; thus, these non-coding RNAs can modulate arterial heterogeneity. The identification of an artery-specific miRNA signature would be promising in the therapy of CVDs, especially in patients who are frail and elderly. In the present review, we will provide a concise description of the arterial tree heterogeneity on a structural and cellular basis, mainly in the pathological context. Secondly, we will address the miRNA potential as crucial mediators of arterial heterogeneity, focusing on the abdominal aorta and femoral artery, with the final goal of strengthening the search for more targeted therapies in CVDs and stratification approaches in patients who are frail and elderly.

Caveolin-1 in situ expression in glomerular and peritubular capillaries as a marker of ultrastructural progression and severity of renal thrombotic microangiopathy.

BACKGROUND: Thrombotic microangiopathy is a severe and potentially life-threatening condition inducing severe endothelial injury in many organs, particularly native and transplanted kidneys. Current pathological studies by our group have identified the use of Caveolin-1 immunohistochemistry as a potential marker of endothelial damage and progression degree of thrombotic microangiopathy. The aim of the present work was to evaluate Caveolin-1 as a marker of severity in thrombotic microangiopathy kidney disease, according to the ultrastructural progression of the disease evaluated by transmission electron microscopy. MATERIALS AND METHODS: Twenty-nine patients (17 non-transplanted and 12 transplanted) were retrospectively selected, biopsied for suspected or histologically-confirmed thrombotic microangiopathy. Transmission electron microscopy was performed in all cases, and an ultrastructural score of thrombotic microangiopathy-related glomerular disease was assessed (from 0 to 3+). Immunohistochemistry for Caveolin-1 was automatically performed. RESULTS: The mean percentage of Caveolin-1-positive glomerular capillaries was 53.2 ± 40.6% and 28.0 ± 42.8% in the active thrombotic microangiopathy versus previous thrombotic microangiopathy cases (p = 0.085), considering both native and transplanted kidneys. The presence of progressive disease correlated with diffuse Caveolin-1 immunoreactivity (p = 0.031), and ultrastructural score correlated with glomerular Caveolin-1 positivity, progressively increasing from 22.5% of the Score 0 group to 95.5% of the Score 3 group (p = 0.036). DISCUSSION: Caveolin-1 proved to be a very useful marker of early endothelial damage in the course of thrombotic microangiopathy for both native and transplanted kidneys, therefore worth considering in routine practice. Diffuse glomerular Caveolin-1 immunoreactivity correlates with the severity of the thrombotic disease and it can appear very early, even before ultrastructurally evident endothelial damage.

The PPAR-γ Agonist Pioglitazone Modulates Proliferation and Migration in HUVEC, HAOSMC and Human Arteriovenous Fistula-Derived Cells.

The failure of arteriovenous fistulas (AVFs) following intimal hyperplasia (IH) increases morbidity and mortality rates in patients undergoing hemodialysis for chronic kidney disease. The peroxisome-proliferator associated receptor (PPAR-γ) may be a therapeutic target in IH regulation. In the present study, we investigated PPAR-γ expression and tested the effect of pioglitazone, a PPAR-γ agonist, in different cell types involved in IH. As cell models, we used Human Endothelial Umbilical Vein Cells (HUVEC), Human Aortic Smooth Muscle Cells (HAOSMC), and AVF cells (AVFCs) isolated from (i) normal veins collected at the first AVF establishment (T0), and (ii) failed AVF with IH (T1). PPAR-γ was downregulated in AVF T1 tissues and cells, in comparison to T0 group. HUVEC, HAOSMC, and AVFC (T0 and T1) proliferation and migration were analyzed after pioglitazone administration, alone or in combination with the PPAR-γ inhibitor, GW9662. Pioglitazone negatively regulated HUVEC and HAOSMC proliferation and migration. The effect was antagonized by GW9662. These data were confirmed in AVFCs T1, where pioglitazone induced PPAR-γ expression and downregulated the invasive genes SLUG, MMP-9, and VIMENTIN. In summary, PPAR-γ modulation may represent a promising strategy to reduce the AVF failure risk by modulating cell proliferation and migration.

Tissue Ki67 proliferative index expression and pathological changes in hemodialysis arteriovenous fistulae: Preliminary single-center results.

BACKGROUND: Arteriovenous fistula (AVF) for hemodialysis integrates outward remodeling with vessel wall thickening in response to drastic hemodynamic changes. Aim of this study is to determine the role of Ki67, a well-established proliferative marker, related to AVF, and its relationship with time-dependent histological morphologic changes. MATERIALS AND METHODS: All patients were enrolled in 1 year and stratified in two groups: (A) pre-dialysis patients submitted to first AVF and (B) patients submitted to revision of AVF. Morphological changes: neo-angiogenesis (NAG), myointimal thickening (MIT), inflammatory infiltrate (IT), and aneurysmatic fistula degeneration (AD). The time of AVF creation was recorded. A biopsy of native vein in Group A and of arterialized vein in Group B was submitted to histological and immunohistochemical (IHC) analysis. IHC for Ki67 was automatically performed in all specimens. Ki67 immunoreactivity was assessed as the mean number of positive cells on several high-power fields, counted in the hot spots. RESULTS: A total of 138 patients were enrolled, 69 (50.0%) Group A and 69 (50.0%) Group B. No NAG or MIT were found in Group A. Seven (10.1%) Group A veins showed a mild MIT. Analyzing the Group B, a moderate-to-severe MIT was present in 35 (50.7%), IT in 19 (27.5%), NAG in 37 (53.6%); AD was present in 10 (14.5%). All AVF of Group B with the exception of one (1.4%) showed a positivity for Ki67, with a mean of 12.31 ± 13.79 positive cells/hot spot (range 0-65). Ki67-immunoreactive cells had a subendothelial localization in 23 (33.3%) cases, a myointimal localization in SMC in 35 (50.7%) cases. The number of positive cells was significantly correlated with subendothelial localization of Ki67 (p = 0.001) and with NA (p = 0.001). CONCLUSIONS: Native veins do not contain cycling cells. In contrast, vascular cell proliferation starts immediately after AVF creation and persists independently of the time the fistula is set up. The amount of proliferating cells is significantly associated with MIT and subendothelial localization of Ki67-immunoreactive cells, thus suggesting a role of Ki-67 index in predicting AVF failure.

Human glial müller and umbilical vein endothelial cell coculture as an in vitro model to investigate retinal oxidative damage. A morphological and molecular assessment.

The aim of this study was to optimize a coculture in vitro model established between the human Müller glial cells and human umbilical vein endothelial cells, mimicking the inner blood-retinal barrier, and to explore its resistance to damage induced by oxidative stress. A spontaneously immortalized human Müller cell line MIO-M1 and human umbilical vein endothelial cells (HUVEC) were plated together at a density ratio 1:1 and maintained up to the 8th passage (p8). The MIO-M1/HUVECs p1 through p8 were treated with increasing concentrations (range 200-800 µM) of H2 O2 to evaluate oxidative stress induced damage and comparing data with single cell cultures. The following features were assayed p1 through p8: doubling time maintenance, cell viability using MTS assay, ultrastructure of cell-cell contacts, immunofluorescence for Vimentin and GFAP, molecular biology (q-PCR) for GFAP and CD31 mRNA. MIO-M1/HUVECs cocultures maintained distinct cell cytotype up to p8 as shown by flow cytometry analysis, without evidence of cross activation, displaying cell-cell tight junctions mimicking those found in human retina, only acquiring a slight resistance to oxidative stress induction over the passages. This MIO-M1/HUVECs coculture represents a simple, reproducible and affordable model for in vitro studies on oxidative stress-induced retinal damages.

The Characteristics and Survival Potential Under Sub-lethal Stress of Mesenchymal Stromal/Stem Cells Isolated from the Human Vascular Wall.

Mesenchymal stromal/stem cells (MSCs) have been identified in multiple human tissues, including the vascular wall. High proliferative potential, multilineage, and immunomodulatory properties make vascular MSCs promising candidates for regenerative medicine. Indeed, their location is strategic for controlling vascular and extra-vascular tissue homeostasis. However, the clinical application of MSCs, and in particular vascular MSCs, is still challenging. Current studies are focused on developing strategies to improve MSC therapeutic applications, like priming MSCs with stress conditions (hypoxia, nutrient deprivation) to achieve a higher therapeutic potential. The goal of the present study is to review the main findings regarding the MSCs isolated from the human vascular wall. Further, the main priming strategies tested on MSCs from different sources are reported, together with the experience on vascular MSCs isolated from healthy cryopreserved and pathological arteries. Stress induction can be a priming approach able to improve MSC effectiveness through several mechanisms that are discussed in this review. Nevertheless, these issues have not been completely explored in vascular MSCs and potential side effects need to be investigated.

Trophism and Homeostasis of Liver Sinusoidal Endothelial Graft Cells during Preservation, with and without Hypothermic Oxygenated Perfusion.

The aim of the present study was to evaluate the homeostasis and trophism of liver sinusoidal endothelial cells (LSECs) in vivo in different stages of liver graft donation, in order to understand the effects of graft ischemia and perfusion on LSEC activity in liver grafts. Special attention was paid to grafts that underwent hypothermic oxygenated perfusion (HOPE). Forty-seven donors were prospectively enrolled, and two distinct biopsies were performed in each case: one allocation biopsy (at the stage of organ allocation) and one post-perfusion biopsy, performed after graft implant in the recipients. In all biopsies, immunohistochemistry and RT-PCR analyses were carried out for the endothelial markers CD34, ERG, Nestin, and VEGFR-2. We observed an increase in CD34 immunoreactivity in LSEC during the whole preservation/perfusion period (p < 0.001). Nestin and ERG expression was low in allocation biopsies, but increased in post-perfusion biopsies, in both immunohistochemistry and RT-PCR (p < 0.001). An inverse correlation was observed between ERG positivity and donor age. Our results indicate that LSEC trophism is severely depressed in liver grafts, but it is restored after reperfusion in standard conditions. The execution of HOPE seems to improve this recovery, confirming the effectiveness of this machine perfusion technique in restoring endothelial functions.

Impact of Freeze-Drying on Cord Blood (CB), Serum (S), and Platelet-Rich Plasma (CB-PRP) Preparations on Growth Factor Content and In Vitro Cell Wound Healing.

Blood-based preparations are used in clinical practice for the treatment of several eye disorders. The aim of this study is to analyze the effect of freeze-drying blood-based preparations on the levels of growth factors and wound healing behaviors in an in vitro model. Platelet-rich plasma (PRP) and serum (S) preparations from the same Cord Blood (CB) sample, prepared in both fresh frozen (FF) and freeze-dried (FD) forms (and then reconstituted), were analyzed for EGF and BDNF content (ELISA Quantikine kit). The human MIO-M1 glial cell line (Moorfield/Institute of Ophthalmology, London, UK) was incubated with FF and FD products and evaluated for cell migration with scratch-induced wounding (IncuCyte S3 Essen BioScience), proliferation with cyclin A2 and D1 gene expression, and activation with vimentin and GFAP gene expression. The FF and FD forms showed similar concentrations of EGF and BDNF in both the S and PRP preparations. The wound healing assay showed no significant difference between the FF and FD forms for both S and PRP. Additionally, cell migration, proliferation, and activation did not appear to change in the FD forms compared to the FF ones. Our study showed that reconstituted FD products maintained the growth factor concentrations and biological properties of FF products and could be used as a functional treatment option.

Epigallocatechin-3-Gallate (EGCG) Mitigates Endothelial and Circulating Cells Alterations Following PLLA Electrospun Mat Placement.

BACKGROUND: Synthetic vascular graft calcification is a serious complication of graft placement. Here, we analysed migration and osteogenic genes of human umbilical vein endothelial cells (HUVEC) cultured with a poly-L-lactic acid (PLLA) electrospun mat. The role of epigallo-catechin-3-gallate (EGCG) in pathogenic processes involving HUVEC and peripheral blood mononuclear cells (PBMCs) was also tested. METHODS: HUVEC were cultured in indirect contact with PLLA for 48 h, with or without EGCG, and processed for mRNA expression. HUVEC proliferation, migration and osteogenic differentiation were evaluated after EGCG treatment. EGCG was also administrated to human PBMCs, to analyse proliferation and migration toward HUVEC cultured with PLLA. RESULTS: HUVEC cultured with PLLA exhibited increased expression of SLUG, VIMENTIN, MMP-9 (migration, vascular remodelling) and RUNX-2 (osteogenic transcription factor). EGCG at 25 µM significantly reduced HUVEC migration, osteogenic differentiation, without affecting cell viability, and mitigated PLLA influence on SLUG, MMP-9, VIMENTIN and RUNX-2 expression. EGCG affected PBMC proliferation and migration toward PLLA in a transwell co-culture system with HUVEC. CONCLUSION: Our study suggests the pro-calcific effect of PLLA, proposing EGCG as an anti-inflammatory modulatory approach. Research efforts need to deepen PLLA-vascular wall interactions for preventing vascular graft failure.

Phenotypic, morphological, and metabolic characterization of vascular-spheres from human vascular mesenchymal stem cells.

The ability to form spheroids under non-adherent conditions is a well-known property of human mesenchymal stem cells (hMSCs), in addition to stemness and multilineage differentiation features. In the present study, we tested the ability of hMSCs isolated from the vascular wall (hVW-MSCs) to grow as spheres, and provide a characterization of this 3D model. hVW-MSCs were isolated from femoral arteries through enzymatic digestion. Spheres were obtained using ultra-low attachment and hanging drop methods. Immunophenotype and pluripotent genes (SOX-2, OCT-4, NANOG) were analyzed by immunocytochemistry and real-time PCR, respectively. Spheres histological and ultrastructural architecture were examined. Cell viability and proliferative capacity were measured using LIVE/DEATH assay and ki-67 proliferation marker. Metabolomic profile was obtained with liquid chromatography-mass spectrometry. In 2D, hVW-MSCs were spindle-shaped, expressed mesenchymal antigens, and displayed mesengenic potential. 3D cultures of hVW-MSCs were CD44+ , CD105low , CD90low , exhibited a low propensity to enter the cell cycle as indicated by low percentage of ki-67 expression and accumulated intermediate metabolites pointing to slowed metabolism. The 3D model of hVW-MSCs exhibits stemness, dormancy and slow metabolism, typically observed in stem cell niches. This culture strategy can represent an accurate model to investigate hMSCs features for future clinical applications in the vascular field.

Adventitial Microcirculation Is a Major Target of SARS-CoV-2-Mediated Vascular Inflammation.

We report the case of a 77-year-old woman affected by coronavirus disease-19 (COVID-19) who developed an occlusive arterial disease of the lower limb requiring a left leg amputation. We studied the mechanisms of vascular damage by SARS-CoV-2 by means of a comprehensive multi-technique in situ analysis on the diseased popliteal arterial district, including immunohistochemistry (IHC), transmission electron microscopy (TEM) and miRNA analysis. At histological analyses, we observed a lymphocytic inflammatory infiltrate, oedema and endothelialitis of adventitial vasa vasorum while the media was normal and the intima had only minor changes. The vasa vasorum expressed the ACE2 receptor and factor VIII; compared with the controls, VEGFR2 staining was reduced. TEM analyses showed endothelial injury and numerous Weibel-Palade bodies in the cytoplasm. No coronavirus particle was seen. IL-6 protein and mRNA, together with miR-155-5p and miRs-27a-5p, which can target IL-6, were significantly increased compared with that in the controls. Our case report suggests an involvement of adventitial artery microcirculation by inflammation in the course of COVID-19. Without evident signs of current infection by SARS-CoV-2, endothelial cells show a spectrum of structural and functional alterations that can fuel the cardiovascular complications observed in people infected with SARS-CoV-2.

ETS-Related Gene Expression in Healthy Femoral Arteries With Focal Calcifications.

Bone development-related genes are enriched in healthy femoral arteries, which are more prone to calcification, as documented by the predominance of fibrocalcific plaques at the femoral location. We undertook a prospective histological study on the presence of calcifications in normal femoral arteries collected from donors. Since endothelial-to-mesenchymal transition (EndMT) participates in vascular remodeling, immunohistochemical (IHC) and molecular markers of EndMT and chondro-osteogenic differentiation were assessed. Transmission electron microscopy (TEM) was used to describe calcification at its inception. Two hundred and fourteen femoral arteries were enrolled. The mean age of the donors was 39.9 ± 12.9 years; male gender prevailed (M: 128). Histology showed a normal architecture; calcifications were found in 52 (24.3%) cases, without correlations with cardiovascular risk factors. Calcifications were seen on or just beneath the inner elastic lamina (IEL). At IHC, SLUG was increasingly expressed in the wall of focally calcified femoral arteries (FCFA). ETS-related gene (ERG), SLUG, CD44, and SOX-9 were positive in calcifications. RT-PCR showed increased levels of BPM-2, RUNX-2, alkaline phosphatase, and osteocalcin osteogenic transcripts and increased expression of the chondrogenic marker, SOX-9, in FCFA. TEM documented osteoblast-like cells adjacent to the IEL, releasing calcifying vesicles from the cell membrane. The vesicles were embedded in a proteoglycan-rich matrix and were entrapped in IEL fenestrations. In this study, ERG- and CD44-positive cell populations were found in the context of increased SLUG expression, thus supporting the participation of EndMT in FCFA; the increased transcript expression of osteochondrogenic markers, particularly SOX-9, reinforced the view that EndMT, osteochondrogenesis, and neoangiogenesis interact in the process of arterial calcification. Given its role as a transcription factor in the regulation of endothelial homeostasis, arterial ERG expression can be a clue of endothelial dysregulation and changes in IEL organization which can ultimately hinder calcifying vesicle diffusion through the IEL fenestrae. These results may have a broader implication for understanding arterial calcification within a disease context.

MicroRNA profiles of human peripheral arteries and abdominal aorta in normal conditions: MicroRNAs-27a-5p, -139-5p and -155-5p emerge and in atheroma too.

Atherosclerosis may starts early in life and each artery has peculiar characteristics likely affecting atherogenesis. The primary objective of the work was to underpin the microRNA (miR)-profiling differences in human normal femoral, abdominal aortic, and carotid arteries. The secondary aim was to investigate if those identified miRs, differently expressed in normal conditions, may also have a role in atherosclerotic arteries at adult ages. MiR-profiles were performed on normal tissues, revealing that aorta and carotid arteries are more similar than femoral arteries. MiRs emerging from profiling comparisons, i.e., miR-155-5p, -27a-5p, and -139-5p, were subjected to validation by RT-qPCR in normal arteries and also in pathological/atheroma counterparts, considering all the available 20 artery specimens. The three miRs were confirmed to be differentially expressed in normal femoral vs aorta/carotid arteries. Differential expression of those miRs was also observed in atherosclerotic arteries, together with some miR-target proteins, such as vimentin, CD44, E-cadherin and an additional marker SLUG. The different expression of miRs and targets/markers suggests that aorta/carotid and femoral arteries differently activate molecular drivers of pathological condition, thus conditioning the morphology of atheroma in adult life and likely suggesting the future use of artery-specific treatment to counteract atherosclerosis.

Involvement of miR-30a-5p and miR-30d in Endothelial to Mesenchymal Transition and Early Osteogenic Commitment under Inflammatory Stress in HUVEC.

The endothelial to mesenchymal transition (End-MT) can be associated with vascular calcification, by providing mesengenic progenitors. In this study, we investigated a link between End-MT and the osteogenic process and explored the involvement of miR-30a-5p and miR-30d as potential regulators of these processes. End-MT was induced in Human Umbilical Vein Endothelial Cells (HUVEC) through transforming growth factor-ß1 (TGF-ß1), TGFß-3 and tumor necrosis factor-α (TNF-α), for 24 h and 6 days. End-MT mediators, mesenchymal and osteo/chondrogenic markers were analyzed through Real-Time PCR, immunofluorescence, flow cytometry and Western Blot. miR-30a-5p and miR-30d over-expression was carried out in HUVEC to explore their effects on End-MT and osteogenic differentiation. HUVEC at 24 h and 6 days gained mesenchymal morphology markers, including matrix metalloproteinase 9 (MMP-9), SLUG, VIMENTIN and α-smooth muscle actin (α-SMA), and a significant migratory potential, notably with TNF-α. After 6 days, the osteo/chondrogenic markers runt-related transcription factor 2 (RUNX-2) and SRY box transcription factor 9 (SOX-9) were upregulated. At this time point, miR-30a-5p and miR-30d decreased. Over-expression of miR-30a-5p and miR-30d affected End-MT mediators and the osteogenic potency in HUVEC, by reducing SLUG, VIMENTIN and RUNX-2. Our data suggest that End-MT represents a key link between inflammation and vascular calcification. Further, miR-30a-5p and miR-30d can regulate both the End-MT and the osteogenic processes, prompting future studies for exploring their potential use as therapeutic targets or biomarkers in vascular diseases.

The carotid plaque as paradigmatic case of site-specific acceleration of aging process: The microRNAs and the inflammaging contribution.

Atherosclerosis is considered a chronic inflammatory disease of arteries associated with the aging process. Many risk factors have been identified and they are mainly related to life-styles, gene-environment interactions and socioeconomic status. Carotid and coronary artery diseases are the two major atherosclerotic conditions, being the primary cause of stroke and heart attack, respectively. Nevertheless, carotid plaque assumes particular aspects not only for the specific molecular mechanisms, but also for the types of atheroma which may be associated with a better or a worst prognosis. The identification of circulating blood biomarkers able to distinguish carotid plaque types (stable or vulnerable) is a crucial step for the improvement of adequate therapeutic approaches avoiding or delaying endarterectomy in the oldest old individuals (> 80 years), a population predicted to growth in the next years. The review highlights the most recent knowledge on carotid plaque molecular mechanisms, focusing on microRNAs (miRs), as a site-specific accelerated aging within the conceptual framework of Geroscience for new affordable therapies.

Effects of Cord Blood Serum (CBS) on viability of retinal Müller glial cells under in vitro injury.

Oxidative stress and inflammation determine retinal ganglion cell degeneration, leading to retinal impairment and vision loss. Müller glial cells regulate retinal repair under injury, through gliosis. Meanwhile, reactive gliosis can turn in pathological effects, contributing to neurodegeneration. In the present study, we tested whether Cord Blood Serum (CBS), rich of growth factors, might improve the viability of Müller cells under in vitro damage. BDNF, NGF, TGF-α, GDNF and EGF levels were measured in CBS samples by Human Magnetic Luminex Assay. CBS effects were evaluated on rat (rMC-1) and human (MIO-M1) Müller cells, under H2O2 and IL-1ß damage. Cells grown with FBS or CBS both at 5% were exposed to stress and analyzed in terms of cell viability, GFAP, IL-6 and TNF-α expression. CBS was also administrated after treatment with K252a, inhibitor of the neurotrophin receptor Trk. Cell viability of rMC-1 and MIO-M1 resulted significantly improved when pretreated with CBS and exposed to H2O2 and IL-1ß, in comparison to the standard culture with FBS. Accordingly, the gliosis marker GFAP resulted down-regulated following CBS priming. In parallel, we observed a lower expression of the inflammatory mediators in rMC-1 (TNF-α) and MIO-M1 (IL-6, TNF- α), especially in presence of inflammatory damage. Trk inhibition through K252a administration impaired the effects of CBS under stress conditions on MIO-M1 and rMC-1 viability, not significantly different from FBS condition. CBS is enriched with neurotrophins and its administration to rMC-1 and MIO-M1 attenuates the cytotoxic effects of H2O2 and IL-1ß. Moreover, the decrease of the main markers of gliosis and inflammation suggests a promising use of CBS for neuroprotection aims. This study is a preliminary basis that prompts future investigations to deeply explore and confirm the CBS potential.

Pharmacological (or Synthetic) and Nutritional Agonists of PPAR-γ as Candidates for Cytokine Storm Modulation in COVID-19 Disease.

The cytokine storm is an abnormal production of inflammatory cytokines, due to the over-activation of the innate immune response. This mechanism has been recognized as a critical mediator of influenza-induced lung disease, and it could be pivotal for COVID-19 infections. Thus, an immunomodulatory approach targeting the over-production of cytokines could be proposed for viral aggressive pulmonary disease treatment. In this regard, the peroxisome proliferator-activated receptor (PPAR)-γ, a member of the PPAR transcription factor family, could represent a potential target. Beside the well-known regulatory role on lipid and glucose metabolism, PPAR-γ also represses the inflammatory process. Similarly, the PPAR-γ agonist thiazolidinediones (TZDs), like pioglitazone, are anti-inflammatory drugs with ameliorating effects on severe viral pneumonia. In addition to the pharmacological agonists, also nutritional ligands of PPAR-γ, like curcuma, lemongrass, and pomegranate, possess anti-inflammatory properties through PPAR-γ activation. Here, we review the main synthetic and nutritional PPAR-γ ligands, proposing a dual approach based on the strengthening of the immune system using pharmacological and dietary strategies as an attempt to prevent/treat cytokine storm in the case of coronavirus infection.

Different Drugs Effect on Mesenchymal Stem Cells Isolated From Abdominal Aortic Aneurysm.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a progressive dilation of the aortic wall, determined by the unbalanced activity of matrix metalloproteinase (MMPs). In vitro and in vivo studies support the pivotal role of MMP-9 to AAA pathogenesis. In our experience, we elucidated the expression of MMP-9 in an ex vivo model of human mesenchymal stem cells isolated from AAA specimen (AAA-MSCs). Thus, MMP-9 inhibition could be an attractive therapeutic strategy for inhibiting AAA degeneration and rupture. Our study was aimed at testing the effect of 3 different drugs (pioglitazone, doxycycline, simvastatin) on MMP-9 and peroxisome proliferator-activated receptor (PPAR)-γ expression in AAA-MSCs. METHODS: Aneurysmal aortic wall segments were taken from AAA patients after the open surgical treatment. MSCs were isolated from AAA (n = 20) tissues through enzymatic digestion. AAA-MSCs were exposed to different doses of pioglitazone (5-10-25 µM), doxycycline (10-25 µM), and simvastatin (10 µM) for 24 h. The effect of each drug was evaluated in terms of cell survival, by crystal violet stain. MMP-9 and PPAR-γ mRNA were analyzed using real-time PCR. RESULTS: AAA-MSCs were not affected by the exposure to the selected drugs, as shown by the analysis of cell viability. Interestingly, MMP-9 mRNA resulted significantly decreased after each treatment, recording a downregulation of 50% in presence of pioglitazone, 90% with doxycycline, and 40% with exposed to simvastatin, in comparison to untreated cells. We further analyzed the expression of PPAR-γ, target of pioglitazone, observing an upregulation in exposed AAA-MSCs to controls. CONCLUSIONS: Our data support the potential therapeutic effect of pioglitazone, doxycycline, and simvastatin on AAA by reducing the MMP-9 expression in a patient-specific model (AAA-MSCs). In addition, pioglitazone drives the increase of PPAR-G, another promising target for AAA therapy. Further studies are necessary to elucidate the mechanism driving this inhibitory pathway, which can reduces the mortality risk associated with AAA rupture.

Different histological types of active intraplaque calcification underlie alternative miRNA-mRNA axes in carotid atherosclerotic disease.

Arterial calcification is an actively regulated process, with different morphological manifestations. Micro-RNAs emerged as potential regulators of vascular calcification; they may become novel diagnostic tools and be used for a finest staging of the carotid plaque progression. The present study aimed at characterizing the different miRNA-mRNA axes in carotid plaques according to their histological patterns of calcification. Histopathological analysis was performed on 124 retrospective carotid plaques, with clinical data and preoperatory angio-CT. miRNA analysis was carried out with microfluidic cards. Real-time PCR was performed for selected miRNAs validation and for RUNX-2 and SOX-9 mRNA levels. CD31, CD68, SMA, and SOX-9 were analyzed by immunohistochemistry. miRNA levels on HUVEC cells were analyzed for confirming results under in vitro osteogenic conditions. Histopathological analysis revealed two main calcification subtypes of plaques: calcific cores (CC) and protruding nodules (PN). miRNA array and PCR validation of miR-1275, miR-30a-5p, and miR-30d indicated a significant upregulation of miR-30a-5p and miR-30d in the PN plaques. Likewise, the miRNA targets RUNX-2 and SOX-9 resulted poorly expressed in PN plaques. The inverse correlation between miRNA and RUNX-2 levels was confirmed on osteogenic-differentiated HUVEC. miR-30a-5p and miR-30d directly correlated with calcification extension and thickness at angio-CT imaging. Our study demonstrated the presence of two distinct morphological subtypes of calcification in carotid atheromatous plaques, supported by different miRNA signatures, and by different angio-CT features. These results shed the light on the use of miRNA as novel diagnostic markers, suggestive of plaque evolution.

Biomarkers in Tears and Ocular Surface: A Window for Neurodegenerative Diseases.

OBJECTIVES: The purpose of this review is to briefly outline current scientific evidence on the potential role of tear analysis and ocular surface evaluation in diagnosis and monitoring of neurodegenerative diseases, especially Alzheimer disease, Parkinson disease, and glaucoma. METHODS: A systematic computerized search in the electronic databases PubMed, MEDLINE, and the Cochrane Collaborations was conducted to find eligible articles which their main topic was to investigate the tear and ocular surface in neurodegenerative diseases. After a first screening of titles and abstracts and a full-text review, 26 articles met the inclusion criteria (1 about the neurodegenerative diseases, 3 about the Alzheimer disease, 11 about the Parkinson disease, 11 about glaucoma, and 1 about amyotrophic lateral sclerosis). RESULTS: The ocular surface picture seems to be altered in the setting of neurodegenerative diseases with specific characteristics according to each disease. They seem to be associated with reduced corneal sensitivity and abnormal tear function, and each one presents the expression of specific biomarkers in tears. CONCLUSIONS: The study of tears and ocular surface appears to be a new and noninvasive promising way to assist in the diagnosis and monitoring of neurodegenerative diseases.