Response of Renal Podocytes to Excessive Hydrostatic Pressure: a Pathophysiologic Cascade in a Malignant Hypertension Model.

BACKGROUND/AIMS: Renal injuries induced by increased intra-glomerular pressure coincide with podocyte detachment from the glomerular basement membrane (GBM). In previous studies, it was demonstrated that mesangial cells have a crucial role in the pathogenesis of malignant hypertension. However, the exact pathophysiological cascade responsible for podocyte detachment and its relationship with mesangial cells has not been fully elucidated yet and this was the aim of the current study. METHODS: Rat renal mesangial or podocytes were exposed to high hydrostatic pressure in an in-vitro model of malignant hypertension. The resulted effects on podocyte detachment, apoptosis and expression of podocin and integrinß1 in addition to Angiotensin-II and TGF-ß1 generation were evaluated. To simulate the paracrine effect podocytes were placed in mesangial cell media pre-exposed to pressure, or in media enriched with Angiotensin-II, TGF-ß1 or receptor blockers. RESULTS: High pressure resulted in increased Angiotensin-II levels in mesangial and podocyte cells. Angiotensin-II via the AT1 receptors reduced podocin expression and integrinß1, culminating in detachment of both viable and apoptotic podocytes. Mesangial cells exposed to pressure had a greater increase in Angiotensin-II than pressure-exposed podocytes. The massively increased concentration of Angiotensin-II by mesangial cells, together with increased TGF-ß1 production, resulted in increased apoptosis and detachment of non-viable apoptotic podocytes. Unlike the direct effect of pressure on podocytes, the mesangial mediated effects were not related to changes in adhesion proteins expression. CONCLUSIONS: Hypertension induces podocyte detachment by autocrine and paracrine effects. In a direct response to pressure, podocytes increase Angiotensin-II levels. This leads, via AT1 receptors, to structural changes in adhesion proteins, culminating in viable podocyte detachment. Paracrine effects of hypertension, mediated by mesangial cells, lead to higher levels of both Angiotensin-II and TGF-ß1, culminating in apoptosis and detachment of non-viable podocytes.

Directionality of noncoding human RNAs: how to avoid artifacts.

Inactivation of tumor suppressor and metastasis suppressor genes via epigenetic silencing is a frequent event in human cancers. Recent work has shown new mechanisms of epigenetic silencing, based on the occurrence of long noncoding promoter-spanning antisense and/or sense RNAs (lncRNAs), which constitute part of chromatin silencing complexes. Using reverse transcription polymerase chain reaction (RT-PCR), we have started to scan "triple negative" and Her2-overexpressing breast cancer cell lines for directional/bidirectional transcription through promoters of tumor suppressor and metastasis suppressor genes known to be epigenetically silenced in vivo. Surprisingly, we found that RT-PCR-amplified products were obtained at high frequency in the absence of exogenous primers. These amplified products resulted from RT priming via transcripts originating from promoter or upstream spanning regions. Consequently, this priming overruled directionality determination and led to false detection-identification of such lncRNAs. We show that this prevalent "no primer" artifact can be eliminated by treating the RNA preparations with periodate, performing RT reactions at highly elevated temperatures, or a combination of both. These experimental improvements enabled determination of the presence and directionality of individual promoter-spanning long noncoding RNAs with certainty. Examples for the BRMS1 metastasis suppressor gene, as well as RAR-ß2 and CST6 human tumor suppressor genes, in breast carcinoma cell lines are presented.

Effects of Hyperbaric Oxygen Therapy on Mitochondrial Respiration and Physical Performance in Middle-Aged Athletes: A Blinded, Randomized Controlled Trial.

INTRODUCTION: Hyperbaric oxygen therapy (HBOT) has been used to increase endurance performance but has yet to be evaluated in placebo-controlled clinical trials. The current study aimed to evaluate the effect of an intermittent HBOT protocol on maximal physical performance and mitochondrial function in middle-aged master athletes. METHODS: A double-blind, randomized, placebo-controlled study on 37 healthy middle-aged (40-50) master athletes was performed between 2018 and 2020. The subjects were exposed to 40 repeated sessions of either HBOT [two absolute atmospheres (ATA), breathing 100% oxygen for 1 h] or SHAM (1.02ATA, breathing air for 1 h). RESULTS: Out of 37 athletes, 16 HBOT and 15 SHAM allocated athletes were included in the final analysis. Following HBOT, there was a significant increase in the maximal oxygen consumption (VO2Max) (p = 0.010, effect size(es) = 0.989) and in the oxygen consumption measured at the anaerobic threshold (VO2AT)(es = 0.837) compared to the SHAM group. Following HBOT, there were significant increases in both maximal oxygen phosphorylation capacity (es = 1.085, p = 0.04), maximal uncoupled capacity (es = 0.956, p = 0.02) and mitochondrial mass marker MTG (p = 0.0002) compared to the SHAM sessions. CONCLUSION: HBOT enhances physical performance in healthy middle-age master athletes, including VO2max, power and VO2AT. The mechanisms may be related to significant improvements in mitochondrial respiration and increased mitochondrial mass. Trial Registration ClinicalTrials.gov Identifier: https://clinicaltrials.gov/ct2/show/NCT03524989 (May 15, 2018).

Association of Variants in TMEM45A With Keratoglobus.

IMPORTANCE: Keratoglobus is a rare corneal disorder characterized by generalized thinning and globular protrusion of the cornea. Affected individuals typically have significantly decreased vision and are at risk of corneal perforation. The genetic basis and inheritance pattern of isolated congenital keratoglobus are currently unknown. OBJECTIVE: To identify the genetic basis of isolated congenital keratoglobus. DESIGN, SETTING, AND PARTICIPANTS: This case series and molecular analysis studied 3 unrelated nonconsanguineous families with keratoglobus at a medical center in Israel. Data were collected from June 2019 to March 2021 and analyzed during the same period. EXPOSURES: Whole-exome sequencing and direct Sanger sequencing, expression analysis by real-time polymerase chain reaction, splice-site variant analysis, immunohistochemical staining, and histological evaluation of a knockout mouse model. MAIN OUTCOMES AND MEASURE: Molecular characteristics associated with keratoglobus. RESULTS: Four pediatric patients (3 male individuals) from 3 families had clinical findings consistent with keratoglobus. These included globular protrusion, corneal thinning more prominent at the periphery, and high astigmatism. Truncating and splice site variants were identified in the TMEM45A gene, which fully segregate with the disorder. All affected individuals were homozygous or compound heterozygous for variants in the TMEM45A gene, while unaffected family members were heterozygous carriers. Expression analysis in healthy controls showed that TMEM45A was expressed 23 times higher in the human cornea compared with peripheral blood. Immunohistochemical staining of the TMEM45A protein in normal corneas confirmed its expression in the corneal stroma and epithelium. A TMEM45A knockout mouse model showed structural features consistent with keratoglobus. CONCLUSIONS AND RELEVANCE: Expression of TMEM45A has been previously shown to result in upregulation of extracellular matrix components and fibrosis. These results suggest that isolated congenital keratoglobus is an autosomal recessively inherited disorder associated with variants in the TMEM45A gene.

The effect of hyperbaric oxygen therapy on the pathophysiology of skin aging: a prospective clinical trial.

INTRODUCTION: Skin biopsies can be used to evaluate physiological effects of aging targeted intervention at the tissue/cellular levels. Recent clinical trials have shown that hyperbaric oxygen therapy (HBOT) can target aging hallmarks, including telomere shortening, senescent cells clearance and angiogenesis. The aim of this study was to evaluate the effects of HBOT on the skin of a normal, non-pathological, aging population. METHODS: The study was performed as a prospective clinical trial. After signing informed consent and undergoing baseline evaluations, the subjects were assigned to a three-month control period followed by three months of HBOT daily sessions. Skin biopsies were taken at baseline, after three months of no intervention (control) and 1-2 weeks following the last HBOT session. Trichrome, Orecin, lipofuscin and CD31 staining were used to evaluate collagen fibers, elastic fibers, senescent cells and blood vessels, respectively. RESULTS: Out of the cohort of 70 participants in the normal aging population study, thirteen male patients (age 68.07±2.5y) gave consent for repeated skin biopsies. Following HBOT, there was a significant increase in collagen density (p<0.001, effect size(es)=1.10), elastic fiber length (p<0.0001, es=2.71) and the number of blood vessels (p=0.02, es=1.00). There was a significant decrease in fiber fragmentation (p=0.012) and in tissue senescent cells (p=0.03, es=0.84) post-HBOT. No changes were noted in elastic fiber density or thickness. CONCLUSIONS: The study indicates, for the first time in humans, that HBOT can significantly modulate the pathophysiology of the skin aging in a healthy aging population. The demonstrated mechanisms include angiogenesis and senescent cell clearance.

Decoding pooled RNAi screens by means of barcode tiling arrays.

BACKGROUND: RNAi screens via pooled short hairpin RNAs (shRNAs) have recently become a powerful tool for the identification of essential genes in mammalian cells. In the past years, several pooled large-scale shRNA screens have identified a variety of genes involved in cancer cell proliferation. All of those studies employed microarray analysis, utilizing either the shRNA's half hairpin sequence or an additional shRNA-associated 60 nt barcode sequence as a molecular tag. Here we describe a novel method to decode pooled RNAi screens, namely barcode tiling array analysis, and demonstrate how this approach can be used to precisely quantify the abundance of individual shRNAs from a pool. RESULTS: We synthesized DNA microarrays with six overlapping 25 nt long tiling probes complementary to each unique 60 nt molecular barcode sequence associated with every shRNA expression construct. By analyzing dilution series of expression constructs we show how our approach allows quantification of shRNA abundance from a pool and how it clearly outperforms the commonly used analysis via the shRNA's half hairpin sequences. We further demonstrate how barcode tiling arrays can be used to predict anti-proliferative effects of individual shRNAs from pooled negative selection screens. Out of a pool of 305 shRNAs, we identified 28 candidate shRNAs to fully or partially impair the viability of the breast carcinoma cell line MDA-MB-231. Individual validation of a subset of eleven shRNA expression constructs with potential inhibitory, as well as non-inhibitory, effects on the cell line proliferation provides further evidence for the accuracy of the barcode tiling approach. CONCLUSIONS: In summary, we present an improved method for the rapid, quantitative and statistically robust analysis of pooled RNAi screens. Our experimental approach, coupled with commercially available lentiviral vector shRNA libraries, has the potential to greatly facilitate the discovery of putative targets for cancer therapy as well as sensitizers of drug toxicity.

Hyperbaric oxygen therapy increases telomere length and decreases immunosenescence in isolated blood cells: a prospective trial.

INTRODUCTION: Aging is characterized by the progressive loss of physiological capacity. At the cellular level, two key hallmarks of the aging process include telomere length (TL) shortening and cellular senescence. Repeated intermittent hyperoxic exposures, using certain hyperbaric oxygen therapy (HBOT) protocols, can induce regenerative effects which normally occur during hypoxia. The aim of the current study was to evaluate whether HBOT affects TL and senescent cell concentrations in a normal, non-pathological, aging adult population. METHODS: Thirty-five healthy independently living adults, aged 64 and older, were enrolled to receive 60 daily HBOT exposures. Whole blood samples were collected at baseline, at the 30th and 60th session, and 1-2 weeks following the last HBOT session. Peripheral blood mononuclear cells (PBMCs) telomeres length and senescence were assessed. RESULTS: Telomeres length of T helper, T cytotoxic, natural killer and B cells increased significantly by over 20% following HBOT. The most significant change was noticed in B cells which increased at the 30th session, 60th session and post HBOT by 25.68%±40.42 (p=0.007), 29.39%±23.39 (p=0.0001) and 37.63%±52.73 (p=0.007), respectively. There was a significant decrease in the number of senescent T helpers by -37.30%±33.04 post-HBOT (P<0.0001). T-cytotoxic senescent cell percentages decreased significantly by -10.96%±12.59 (p=0.0004) post-HBOT. In conclusion, the study indicates that HBOT may induce significant senolytic effects including significantly increasing telomere length and clearance of senescent cells in the aging populations.

Cognitive enhancement of healthy older adults using hyperbaric oxygen: a randomized controlled trial.

More than half of community-dwelling individuals sixty years and older express concern about declining cognitive abilities. The current study's aim was to evaluate hyperbaric oxygen therapy (HBOT) effect on cognitive functions in healthy aging adults.A randomized controlled clinical trial randomized 63 healthy adults (>64) either to HBOT(n=33) or control arms(n=30) for three months. Primary endpoint included the general cognitive function measured post intervention/control. Cerebral blood flow (CBF) was evaluated by perfusion magnetic resonance imaging.There was a significant group-by-time interaction in global cognitive function post-HBOT compared to control (p=0.0017). The most striking improvements were in attention (net effect size=0.745) and information processing speed (net effect size=0.788).Voxel-based analysis showed significant cerebral blood flow increases in the HBOT group compared to the control group in the right superior medial frontal gyrus (BA10), right and left supplementary motor area (BA6), right middle frontal gyrus (BA6), left middle frontal gyrus (BA9), left superior frontal gyrus (BA8) and the right superior parietal gyrus (BA7).In this study, HBOT was shown to induce cognitive enhancements in healthy aging adults via mechanisms involving regional changes in CBF. The main improvements include attention, information processing speed and executive functions, which normally decline with aging.

Human mesenchymal stromal cells ameliorate complement induced inflammatory cascade and improve renal functions in a rat model of ischemia-reperfusion induced acute kidney injury.

INTRODUCTION: The primary rational for using mesenchymal stromal cells (MSCs) to rejuvenate damaged tissue is mostly based on their capacity to trans-differentiate and repair injured organs. However, previous studies have demonstrated that MSCs are beneficial even at very early stages, before differentiation and proliferation can be expected. The aim of the current study was to investigate the multifaceted immunological effects of systemically administrating MSCs in the setting of acute kidney injury (AKI) induced by ischemic-reperfusion (I/R). METHODS: A rat model of I/R induced AKI was used. The rats underwent a unilateral nephrectomy with simultaneously clamping the contralateral kidney for 60 minutes. Four treatment groups received intravenously, increasing doses of human MSCs and after 48 hours, the rats were sacrificed. Blood was taken to evaluate renal functions and to measure systemic inflammatory markers. Kidneys were taken for histopathologic examinations and evaluations of intra-renal complement activation and inflammatory mediators. RESULTS: Renal functions improved in U shaped dose dependent manner. Mean serum creatinine levels were 4.5, 2.9, 2.6, 1.7 and 4.1 mg/dL in I/R + placebo, I/R + 150x103 cells, I/R + 250x103 cells, I/R + 500x103 cells and I/R + 1,000x103 cells respectfully (p-values<0.05). Urea demonstrated consistent results with the same U shape improvement manner. The extensive activation of the complement system was ameliorated in the MSCs treatment groups. In addition, MSCs significantly decreased intra-renal levels of IL-1ß and TNF-α. It should be noted that the highest doses of MSCs induced renal hypoxia, marked by the Hypoxy-probe staining. CONCLUSIONS: The early beneficial effect of MSCs in the setting of AKI may be attributed to their immunomodulatory effects. Safe treatment with MSCs can block the deleterious activation of the complement cascade and alleviate the hazardous inflammatory mediator-related cascade.

The Small Tellurium Compound AS101 Ameliorates Rat Crescentic Glomerulonephritis: Association with Inhibition of Macrophage Caspase-1 Activity via Very Late Antigen-4 Inactivation.

Crescentic glomerulonephritis (CGN) is the most aggressive form of GN and, if untreated, patients can progress to end-stage renal failure within weeks of presentation. The α4ß1 integrin very late antigen-4 (VLA-4) is an adhesion molecule of fundamental importance to the recruitment of leukocytes in inflammation. We addressed the role of VLA-4 in mediating progressive renal injury in a rat model of CGN using a small tellurium compound. AS101 [ammonium trichloro(dioxoethylene-o,o')tellurate]. This compound has been previously shown to uniquely inhibit VLA-4 activity by redox inactivation of adjacent thiols in the exofacial domain of VLA-4. The study shows that administration of AS101 either before or after glomerular basement membrane anti-serum injection ameliorates crescent formation or preserves renal function. This was associated with profound inhibition of critical inflammatory mediators, accompanied by decreased glomerular infiltration of macrophages. Mechanistic studies demonstrated vla-4 inactivation on glomerular macrophages both in vitro and in vivo as well as inhibition of caspase-1 activity. Importantly, this cysteine protease activity modification was dependent on VLA-4 inactivation and was associated with the anti-inflammatory activity of AS101. We propose that inactivation of macrophage VLA-4 by AS101 in vivo results in a decrease of inflammatory cytokines and chemokines produced in the glomeruli of diseased rats, resulting in decreased further macrophage recruitment and decreased extracellular matrix expansion. Thus, AS101, which is currently in clinical trials for other indications, might be beneficial for treatment of CGN.

ER-alpha-cDNA as part of a bicistronic transcript gives rise to high frequency, long term, receptor expressing cell clones.

Within the large group of Estrogen Receptor alpha (ERα)-negative breast cancer patients, there is a subgroup carrying the phenotype ERα(-), PR(-), and Her2(-), named accordingly "Triple-Negative" (TN). Using cell lines derived from this TN group, we wished to establish cell clones, in which ERα is ectopically expressed, forming part of a synthetic lethality screening system. Initially, we generated cell transfectants expressing a mono-cistronic ERα transcription unit, adjacent to a separate dominant selectable marker transcription unit. However, the yield of ERα expressing colonies was rather low (5-12.5%), and only about half of these displayed stable ectopic ERα expression over time. Generation and maintenance of such cell clones under minimal exposure to the ERα ligand, did not improve yield or expression stability. Indeed, other groups have also reported grave difficulties in obtaining ectopic expression of ERα in ERα-deficient breast carcinoma cells. We therefore switched to transfecting these cell lines with pERα-IRES, a plasmid vector encoding a bicistronic translation mRNA template: ERα Open Reading Frame (ORF) being upstream followed by a dominant-positive selectable marker (hygro(R)) ORF, directed for translation from an Internal Ribosome Entry Site (IRES). Through usage of this bicistronic vector linkage system, it was possible to generate a very high yield of ERα expressing cell clones (50-100%). The stability over time of these clones was also somewhat improved, though variations between individual cell clones were evident. Our successful experience with ERα in this system may serve as a paradigm for other genes where ectopic expression meets similar hardships.