Jayachandran, Muthuvel

BACKGROUND: Senescence, a mechanism of cellular aging, which is characterized by irreversible proliferation arrest and a proinflammatory secretory phenotype, has been documented in women with preeclampsia. As cellular senescence can persist and progress, we postulated that it is associated with accelerated aging phenotype and accumulation of comorbidities in women with a history of preeclampsia. METHODS: We included a cohort of women with a history of preeclampsia (n=40) age- and parity-matched to a group of referent women with normotensive pregnancies (n=40). Women with prior major cardiovascular events, neurological, or autoimmune conditions were excluded. We collected urine and blood samples to study markers of aging, data on multimorbidity at the time of enrollment, and prospectively followed them for events over the course of 6 years, on average. RESULTS: Women with a history of preeclampsia exhibited unfavorable aging profiles compared with referent women, including decreased urinary α-Klotho ( P =0.018); increased leptin ( P =0.016) and leptin/adiponectin ratio ( P =0.027), and increased extracellular vesicles positive for tissue factor ( P =0.025). Women with a history of preeclampsia likewise had a higher rate of comorbidities at the time of enrollment ( P =0.003) and had a 4× higher risk of developing major cardiovascular events compared with referent women ( P =0.003). CONCLUSIONS: Our data suggest that a history of preeclampsia is associated with accelerated aging as indicated by senescence marker differences and the accumulation of multimorbidity later in life. Targeting cellular senescence may offer novel, mechanism-based approaches for the diagnosis and treatment of adverse health outcomes in women with a history of preeclampsia.

Objective Activation of brain microvascular endothelium by inflammatory and procoagulant stimuli increases expression of surface cell‐adhesion molecules (CAMs), releases endothelium‐derived microvesicles (MV) into the circulation, and increases permeability of the blood‐brain barrier (BBB). This study evaluated characteristics of MV released from human brain microvascular endothelial cells (HBMEC) following stimulation with proinflammatory or procoagulant stimuli and examined their effect on monolayer permeability. Methods Cultured HBMECs from age‐matched male and female donors were treated for 24 hours with medium supplemented with tumor necrosis factor alpha (TNF α , 20 ng/mL), thrombin (THR, 2 U/mL), or vehicle (control). MVs released into the medium were isolated by differential centrifugation, labeled with annexin V together with antibodies against PECAM‐1, ICAM‐1, E‐selectin, integrin aV, or MCAM, and then analyzed by digital flow cytometry. Permeability (mg/mL FITC‐dextran) of the HBMEC monolayer in response to MV treatment was quantified by fluorescence microplate assay of passage of FITC‐dextran across monolayer cells cultured on microporous supports. Results Fewer specific antigen‐positive MV were released from unstimulated male compared to female HBMECs. TNF a , but not THR induced early‐stage apoptosis; determined by fluorogenic Caspase‐3 assay in the HBMEC monolayers. TNF a induced release of all MV subtypes from male cells, whereas only integrin aV, ICAM‐1 and E‐selectin positive MVs were released from female cells. THR increased release of PECAM‐1, ICAM‐1, and MCAM positive MVs from male cells, but did not increase release of MV in female cells. After 24 hours exposure to MV (2,500 MV/mL) derived from TNF a ‐treated female cells, permeability increased by 17%. Permeabiity was not increased when female cells were exposed to THR‐generated MV. Conclusions These results indicate that: 1) inflammatory and procoagulant stimuli release a different array of MV subtypes from male and female HBMECs; 2) specific stimuli‐derived MVs can modify HMBMEC permeability. Support or Funding Information (Supported by NIH P50 AG44170 and the Mayo Clinic Fnd.).

Objective Preeclampsia (PE) is a hypertensive pregnancy disorder that represents a sex‐specific risk factor for future cardiovascular disease in affected women. This study evaluated the associations of a history of PE, conventional cardiovascular risk factors and vascular activation (defined by activation of blood elements and blood‐borne microvesicles (MV)) with carotid intima‐media thickness (CIMT) in menopausal women. Methods Caucasian women residents of Olmsted County, MN who delivered between 1976 and 1982 were identified from review of their medical records and matched for parity and age at index birth: 40 with a history of preeclampsia (PE) and 40 with a history of normotensive pregnancy (NP). Blood clinical chemistries and 38 measures of intravascular activation including differential cell counts, platelet aggregation and secretion, cell‐cell interactions, and blood‐borne MV were measured in venous blood. CIMT was measured by B‐mode ultrasound. The study was approved by the IRB at Mayo Clinic and all women gave written informed consent. Results Women averaged 60 years of age at the time of testing. CIMT was greater in the PE [0.80 mm (0.75, 0.85); median (25th, 75th percentile)] compared to the NP [0.73 mm (0.70, 0.78)] group (P=0.004). Body mass index, waist circumference, fasting insulin, high‐sensitive C‐reactive protein and present day hypertension were greater in the PE group. After adjusting for potential confounding factors, the odds of having a higher CIMT value was more than 3‐fold higher for women with a history of PE [OR 3.46 (1.38, 8.68); P=0.008]. Principal components (PC) analysis identified 7 components that accounted for 63% of the variability in measures of intravascular activation. The global test for association of intravascular activation with CIMT (P=0.009) was only significant among women with a history of NP. PC#4, which conveyed measures of whole blood platelet aggregation and platelet interactions with granulocytes and monocytes, positively associated with CIMT in the overall group after adjustment for PE status (partial ρ=0.290; P=0.009). PC#6, consisting of measures of platelet activation with platelet count and interactions between monocytes and the vascular endothelium, also showed a significant interaction with pregnancy history (P=0.014). Conclusion A history of preeclampsia accompanied by metabolic and pro‐coagulant/‐inflammatory intravascular cellular activation contributes to progression of CIMT in menopausal women. Support or Funding Information This work was funded by grants from the NIH AG44170, AG034676, UL1 TR000135 and the Mayo Foundation.

The disruption of the cerebral microvascular endothelium and increases in capillary permeability are characteristic of many brain pathologies, including Alzheimer's disease, small vessel disease, ischemia/hypoxia, and hemorrhagic stroke. This study was designed to investigate whether microvesicles (MVs) released from activated human brain microvascular endothelial cells (HBMECs) alter permeability of endothelial monolayers. Male donor HBMECs were cultured to confluence in flasks, then treated for 24 hrs with medium alone (control) or with medium plus either tumor necrosis factor (TNF a , 20 ng/ml), thrombin (THR, 2 U/ml), angiotensin II (AngII, 10 nM), or hydrogen peroxide (H 2 O 2 , 0.8 mM). MVs released into the medium were isolated by differential centrifugation, labeled with annex in V plus antibodies against endothelial adhesion molecules which have modified expression during inflammation (PECAM‐1, ICAM‐1, E‐select in, integrin aV, or MCAM); and then analyzed by FACS. Total number of MVs released (MVs/ml) by each of the agonists (TNF a , 615.8±13.9 MV/μL; THR, 113.8±4.8 MV/μL; and H 2 O 2, 152.8±23.5 MV/μL) were significantly greater (P<0.05) compared the number released from cells treated medium alone (82.2±5.4 MV/μL). Each agonist treatment released distinct types of MV identified by FACS. Permeability (% control) of the HBMEC monolayer assessed using FITC‐dextran transport across the monolayers increased after 90 minute exposure to all agonist treatments: 199.5%, 234.1%, 126.8%, and 588.3% for TNF a , THR, AngII, and H 2 O 2 , respectively. MV derived from the treated cells were labeled with PKH67 and applied to monolayers of (control) HBMECs cultures. Within 30 minutes of exposure punctate structures with PKH67 labeling were observed within the cytoplasm, and after 60 min, similar structures also associated with nuclei and some of which were co‐labeled with the lysosome marker lysotracker red. Co‐localization of labeled MVs with early endosomes, (labeled with anti‐EEA‐1 Ab/Alexa Fluor 647) was absent at these time points. Within these parameters, no differences in vesicle‐trafficking were observed between MVs generated from cells exposed to either medium, TNF a , or H 2 O 2 . These results suggest that treatment of HBMECs with agonists associated with inflammation and thrombosis increase release of distinct MVs which in turn increase paracellular permeability. Capillary endothelial permeability may be disturbed by populations of MVs released from proximal activated cells or MV derived from activated cells and carried in the blood. Support or Funding Information Supported by the Aurora Fnd, NIH HL88988 and Mayo Clinic

AbstractBackgroundPreeclampsia (PE), a pregnancy specific hypertensive disorder, has been associated with elevated risk for strokes, cognitive decline, and smaller brain volumes later in life. As severe PE has been associated with the highest risks for cerebrovascular disease, we hypothesized that circulating extracellular vesicles (EVs) of neurovascular origin will be detectable in women years after severe PE as a marker of persistent neurovascular damage and amyloid‐β.MethodA cohort of 40 women with histories of normotensive pregnancies (control group) and age‐ and parity‐matched to 40 women with history of mild (n = 33) and severe (n = 7) PE were identified using the Rochester Epidemiology Project. Diagnosis of severe PE was ascertained based on clinical criteria (Table). While none of the women had any major cardiovascular events, our previous study of this cohort has demonstrated that total gray matter volumes were smaller in women with a history of preeclampsia and late‐life hypertension compared with the other groups. Blood‐borne EVs derived from neurovascular cellular activation were determined by standardized digital flow cytometry. Plasma concentration of amyloid‐β was measured by ELISA. Differences among the groups were tested by ANOVA, with the least difference test for pos hoc analysis. The association between EVs and MRI brain imaging was assessed by Pearson correlation coefficient.ResultWomen with history of severe PE had a significantly higher concentration of amyloid‐β carrying EVs compared to controls (p = 0.003). EVs positive for the markers of blood‐brain barrier‐ endothelial damage (LDL‐R) and inflammatory coagulation pathway activator (tissue factor), were significantly higher in women with history of severe PE compared to controls (p = 0.008 and p = 0.002, respectively), as well as to the women with history of mild PE. Plasma concentration of total amyloid‐β was also significantly greater in women with history of severe vs. mild PE (p = 0.037) (Table). The number of tissue factor positive EVs was negatively correlated with total gray matter volume (cm3) (p<0.05).ConclusionWomen with a history of severe PE demonstrate elevated levels of markers of neuroinflammation and neurovascular damage, as well as greater amyloid‐β secretion. Excessive inflammation may contribute to previously described brain atrophy in these women.

Anoctamins (ANO) are activated by intracellular Ca2+ and function as phospholipid scramblases and/or Cl- channels. ANO4 is mutated in miniature schnauzers with calcium oxalate stones (CaOx), and ANO4 protein in urinary extracellular vesicles is decreased in human CaOx stone formers. In Drosophila Malpighian tubules (MT), subdued (ANO4-homolog) functions as Ca2+-activated Cl- channel, scramblase, and plays a role in host defense against gram-negative bacteria. Increasing evidence shows the participation of bacteria in urinary stone formation in humans. We used a Drosophila avatar to investigate whether subdued changes CaOx crystallization and if crystallization increases with bacterial infection.Uro:Gal4 flies were crossed with UAS:subdued-RNAi flies to knockdown (KD) subdued in MT principal cells (PC) or with w1118 flies to generate normal controls (WT). F1 generation underwent CaOx crystallization in association or not with uropathogenic E. coli (UPEC) infection. In vivo, flies were fed on diet supplemented with 20mM sodium Ox (NaOx) + UPEC:eGFP for 4 days. For ex vivo assays, dissected MTs were submerged in solution containing 10mM NaOx+ UPEC:eGFP for 90 minutes. subdued-KD in PC reduced subdued mRNA by 56% in MT. Neither crystal number nor size were changed by subdued-KD after prolonged NaOx feeding. However, when UPEC is introduced with the NaOx diet, subdued-KD flies exhibited a large bacterial presence in MT lumen and produced enlarged CaOx crystals compared to WT flies. Similar augmented presence of UPEC in subdued-KD MTs was observed during ex vivo assays. Short-term exposure to NaOx+UPEC resulted in increased number of crystals in subdued-KD MTs compared to WT MTs, but no difference was observed in crystal size.Fly cell atlas ( flycellatlas.org ) revealed that subdued is also found in MT stellate cells (SC; water and Cl- secretion). C724:Gal4>UAS:subdued-RNAi flies were used to assess the effect of subdued-KD in SC. This s ubdued-KD slightly increased bacterial infection but did change CaOx crystallization in MTs after 4 days of NaOx+UPEC feeding.The presence of subdued in PC limits CaOx crystals formation and aggregation through reducing bacterial invasion in the MT lumen. These Drosophila results suggest a role for ANO4 in bacterial-related human and dog kidney stone formation. Future experiments will determine whether other functional aspects of subdued affect CaOx crystallization. U54-DK100227, R01-DK092408, FAPESP (2022/01226-1), Mayo Foundation. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: Low urine pH and volume are established risk factors for uric acid (UA) stone disease (UASD). Renal tubular epithelial cells exposed to an acidic pH and/or UA crystals can shed extracellular vesicles (EVs) into the tubular fluid, and these EVs may be a pathogenic biomarker of UASD. Methods: Urinary EVs bearing UA transporters (SLC2A9, SLC17A3, SLC22A12, SLC5A8, ABCG2, and ZNF365) were quantified in urine from UA stone formers (UASFs), calcium stone formers (CSFs), and age-/sex-matched non-stone formers (NSFs) using a standardized and published method of digital flow cytometry. Results: Urinary pH was lower (p < 0.05) and serum and urinary UA were greater (p < 0.05) in UASFs compared with NSFs. Urinary EVs carrying SLC17A3 and SLC5A8 were lower (p < 0.05) in UASFs compared with NSFs. Urinary EVs bearing SLC2A9, SLC22A12, SLC5A8, ABCG2, and ZNF365 were lower (p < 0.05) in CSFs than UASFs, while excretion of SLC17A3-bearing EVs did not differ between groups. Conclusion: EVs bearing specific UA transporters might contribute to the pathogenesis of UASD and represent non-invasive pathogenic biomarkers for calcium and UA stone risk.

Background and objectives Urinary stone disease has been associated with inflammation, but the specific cell interactions that mediate events remain poorly defined. This study compared calcification and inflammatory cell patterns in kidney tissue from radical nephrectomy specimens of patients without and with a history of urinary stone disease. Design, setting, participants, & measurements Nontumor parenchyma of biobanked radical nephrectomy specimens from age- and sex-matched stone formers ( n =44) and nonstone formers ( n =82) were compared. Calcification was detected by Yasue staining and inflammatory cell populations by immunohistochemistry for CD68 (proinflammatory M1 macrophages), CD163 and CD206 (anti-inflammatory M2 macrophages), CD3 (T lymphocytes), and tryptase (mast cells). Calcifications and inflammatory cells were quantified in cortex and medulla using Image-Pro analysis software. Results Calcification in the medulla of stone formers was higher than in nonstone formers ( P <0.001). M1 macrophages in the cortex and medulla of stone formers were greater than in nonstone formers ( P <0.001), and greater in stone former medulla than stone former cortex ( P =0.02). There were no differences in age, sex, body mass index, tumor characteristics (size, stage, or thrombus), vascular disease status, or eGFR between the groups. M2 macrophages, T lymphocytes, and mast cells did not differ by stone former status. There was a correlation between M1 macrophages and calcification in the medulla of stone formers (rho=0.48; P =0.001) and between M2 macrophages and calcification in the medulla of nonstone formers (rho=0.35; P =0.001). T lymphocytes were correlated with calcification in the cortex of both nonstone formers (rho=0.27; P =0.01) and stone formers (rho=0.42; P =0.004), whereas mast cells and calcification were correlated only in the cortex of stone formers (rho=0.35; P =0.02). Conclusions Higher medullary calcification stimulated accumulation of proinflammatory rather than anti-inflammatory macrophages in stone formers.