Electric cell-substrate impedance sensing has been used to measure transepithelial and transendothelial impedances of cultured cell layers and extract cell parameters such as junctional resistance, cell-substrate separation, and membrane capacitance. Previously, a three-path cell-electrode model comprising two transcellular pathways and one paracellular pathway was developed for the impedance analysis of MDCK cells. By ignoring the resistances of the lateral intercellular spaces, we develop a simplified three-path model for the impedance analysis of epithelial cells and solve the model equations in a closed form. The calculated impedance values obtained from this simplified cell-electrode model at frequencies ranging from 31.25 Hz to 100 kHz agree well with the experimental data obtained from MDCK and OVCA429 cells. We also describe how the change in each model-fitting parameter influences the electrical impedance spectra of MDCK cell layers. By assuming that the junctional resistance is much smaller than the specific impedance through the lateral cell membrane, the simplified three-path model reduces to a two-path model, which can be used for the impedance analysis of endothelial cells and other disk-shaped cells with low junctional resistances. The measured impedance spectra of HUVEC and HaCaT cell monolayers nearly coincide with the impedance data calculated from the two-path model.

Mesenchymal stem cells (MSCs) possess immunomodulatory properties and capacity for endogenous regeneration. Therefore, MSC therapy is a promising treatment strategy for COVID-19. However, the cells cannot stay in the lung long enough to exert their function. The extracellular matrix from porcine bladders (B-ECM) has been shown not only to regulate cellular activities but also to possess immunoregulatory characteristics. Therefore, it can be hypothesized that B-ECM hydrogel could be an excellent scaffold for MSCs to grow and could anchor MSCs long enough in the lung so that they can exhibit their immunomodulatory functions. In this study, ECM degradation products and a co-culture system of MSCs and macrophages were developed to study the immunomodulatory properties of ECM and MSCs under septic conditions. The results showed that B-ECM degradation products could decrease pro-inflammatory and increase anti-inflammatory cytokines from macrophages. In an in vivo mimicking co-culture system, MSCs cultured on B-ECM hydrogel exhibited immunomodulatory properties at both gene and protein levels. Both B-ECM degradation products and MSC conditioned medium supported the wound healing of alveolar epithelial cells. The results from the study could offer a basis for investigation of immunomodulation by ECM and MSCs before conducting in vivo experiments, which could later be applied in regenerative medicine.

Electric Cell-substrate Impedance Sensing (ECIS) is an impedance-based, real-time, and label-free measuring system for monitoring cellular activities in tissue culture. Previously, ECIS wound healing assay has been used to wound cells with high electric current and monitor the subsequent cell migration. In this study, we applied ECIS electric fence (EF) method, an alternative to electrical wounding, to assess the effects of different surface coatings on human keratinocyte (HaCaT) migration. The EF prevents inoculated cells from attaching or migrating to the fenced electrode surface while maintaining the integrity of the surface coating. After the EF is turned off, cells migrate into the cell-free area, and the increase in measured impedance is monitored. We cultured HaCaT cells on gold electrodes without coating or coated with poly-L-lysin (PLL), poly-D-lysine (PDL), or type-I collagen. We quantified migration rates according to the different slopes in the impedance time series. It was observed that either poly-L-lysine (PLL) or poly-D-lysine (PDL) limits cell adhesion and migration rates. Furthermore, the surface charge of the coated substrate in the culture condition positively correlates with the cell adhesion and migration process. Our results indicate that the EF method is useful for determining cell migration rates on specific surface coatings.

Electric cell-substrate impedance sensing (ECIS) has been used as a real-time impedance-based method to quantify cell behavior in tissue culture. The method is capable of measuring both the resistance and capacitance of a cell-covered microelectrode at various AC frequencies. In this study, we demonstrate the application of high-frequency capacitance measurement (f = 40 or 64 kHz) for the sensitive detection of both the micromotion and wound-healing migration of human mesenchymal stem cells (hMSCs). Impedance measurements of cell-covered electrodes upon the challenge of various concentrations of carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), from 0.1 to 30 μM, were conducted using ECIS. FCCP is an uncoupler of mitochondrial oxidative phosphorylation (OXPHOS), thereby reducing mitochondrial ATP production. By numerically analyzing the time-series capacitance data, a dose-dependent decrease in hMSC micromotion and wound-healing migration was observed, and the effect was significantly detected at levels as low as 0.1 μM. While most reported works with ECIS use the resistance/impedance time series, our results suggest the potential use of high-frequency capacitance time series for assessing migratory cell behavior such as micromotion and wound-healing migration.

Background: Chronic kidney disease (CKD) has emerged as an independent risk factor for cerebral microhemorrhages (CMH). We examined molecular mechanisms of CMH formation in mouse models of CKD and hypertension (HTN) using RNA transcriptomics and mTOR complex 1 (mTORC1) inhibition. Methods: Aged (17-month-old) mice were treated with either 0.2% adenine diet to induce CKD, or angiotensin II (ATII) via subcutaneous osmotic pumps to induce HTN and compared with control mice. A subset of CKD mice was treated with rapamycin to inhibit mTORC1 signaling. After 4 weeks, RNA sequencing of mouse brain was done via Illumina NovaSeq 6000 with downstream analysis using Hisat2, Salmon, and DESeq2. Differentially expressed genes (DEGs) and enriched pathways were identified using the MsigDB Hallmark 2020 database. Pathway analysis was further examined using qPCR and immunohistochemistry staining. CMH were quantified using Prussian blue histology. Results: CKD mice had increased CMH number (1.12±0.10 vs 0.85±0.08 per cm 2 in CKD vs control mice, p<0.05), and whole brain RNA-seq showed significant alterations in the mTORC1 pathway, a critical regulator for cell growth and metabolism. Specifically, 18 DEGs were involved in mTORC1 signaling in CKD compared with control animals. qPCR and immunohistochemistry analysis confirmed CKD-associated upregulation of DNA damage-induced transcript 4 (DDIT4), an upstream component of the mTORC1 pathway. ATII-induced HTN more than doubled CMH formation (1.44±0.47 vs. 0.60±0.66 per cm 2 in HTN vs. control mice, p< 0.01), without significant alteration of the mTORC1 pathway. Treatment of CKD mice with the mTOR inhibitor rapamycin significantly reduced CMH burden (24% reduction in CMH number, p<0.05 and 56% reduction in CMH area, p<0.01). Conclusions: Cerebral microhemorrhages were increased in both the CKD and HTN models. However, alteration of brain mTORC1 pathway was observed only in CKD animals, suggesting a specific molecular signature in the CKD brain. Our study supports mTORC1 inhibition as a novel approach to inhibit cerebral microhemorrhage formation in CKD.

Dyslipidemia and insulin resistance are significant adverse outcomes of consuming high-sugar diets. Conversely, dietary fish oil (FO) reduces plasma lipids. Diet-induced dyslipidemia in a rhesus model better approximates the pathophysiology of human metabolic syndrome (MetS) than rodent models. Here, we investigated relationships between metabolic parameters and hypertriglyceridemia in rhesus macaques consuming a high-fructose diet (n = 59) and determined the effects of FO supplementation or RNA interference (RNAi) on plasma ApoC3 and triglyceride (TG) concentrations. Fructose supplementation increased body weight, fasting insulin, leptin, TGs, and large VLDL particles and reduced adiponectin concentrations (all P < 0.001). In multiple regression analyses, increased plasma ApoC3 was the most consistent and significant variable related to diet-induced hypertriglyceridemia. FO supplementation, which attenuated increases of plasma TG and ApoC3 concentrations, reversed fructose-induced shifts of lipoprotein particle size toward IDL and VLDL, a likely mechanism contributing to beneficial metabolic effects, and reduced hepatic expression of genes regulated by the SREBP pathway, particularly acetyl-CoA carboxylase. Furthermore, RNAi-mediated ApoC3 inhibition lowered plasma TG concentrations in animals with diet-induced hypertriglyceridemia. In summary, ApoC3 is an important independent correlate of TG-rich lipoprotein concentrations in rhesus macaques consuming a high-fructose diet. ApoC3 is a promising therapeutic target for hypertriglyceridemia in patients with MetS and diabetes.

MicroRNAs (miRNAs) are important posttranscriptional regulators of metabolism and energy homeostasis. Dysregulation of certain miRNAs in the liver has been shown to contribute to the pathogenesis of Type 2 diabetes (T2D), in part by impairing hepatic insulin sensitivity. By small RNA-sequencing analysis, we identified seven hepatic miRNAs (including miR-29b) that are consistently aberrantly expressed across five different rodent models of metabolic dysfunction that share the feature of insulin resistance (IR). We also showed that hepatic miR-29b exhibits persistent dysregulation during disease progression in a rat model of diabetes, UCD-T2DM. Furthermore, we observed that hepatic levels of miR-29 family members are attenuated by interventions known to improve IR in rodent and rhesus macaque models. To examine the function of the miR-29 family in modulating insulin sensitivity, we used locked nucleic acid (LNA) technology and demonstrated that acute in vivo suppression of the miR-29 family in adult mice leads to significant reduction of fasting blood glucose (in both chow-fed lean and high-fat diet-fed obese mice) and improvement in insulin sensitivity (in chow-fed lean mice). We carried out whole transcriptome studies and uncovered candidate mechanisms, including regulation of DNA methyltransferase 3a (Dnmt3a) and the hormone-encoding gene Energy homeostasis associated (Enho). In sum, we showed that IR/T2D is linked to dysregulation of hepatic miR-29b across numerous models and that acute suppression of the miR-29 family in adult mice leads to improved glycemic control. Future studies should investigate the therapeutic utility of miR-29 suppression in different metabolic disease states.Enho; insulin resistance; liver; microRNA-29 (miR-29); UCD-T2DM.

Background

Chronic kidney disease (CKD) is increasingly recognized as a stroke risk factor, but its exact relationship with cerebrovascular disease is not well-understood. We investigated the development of cerebral small vessel disease using in vivo and in vitro models of CKD.

Methods

CKD was produced in aged C57BL/6J mice using an adenine-induced tubulointerstitial nephritis model. We analyzed brain histology using Prussian blue staining to examine formation of cerebral microhemorrhage (CMH), the hemorrhagic component of small vessel disease and the neuropathological substrate of MRI-demonstrable cerebral microbleeds. In cell culture studies, we examined effects of serum from healthy or CKD patients and gut-derived uremic toxins on brain microvascular endothelial barrier.

Results

CKD was induced in aged C57BL/6J mice with significant increases in both serum creatinine and cystatin C levels (p < 0.0001) without elevation of systolic or diastolic blood pressure. CMH was significantly increased and positively correlated with serum creatinine level (Spearman r = 0.37, p < 0.01). Moreover, CKD significantly increased Iba-1-positive immunoreactivity by 51% (p < 0.001), induced a phenotypic switch from resting to activated microglia, and enhanced fibrinogen extravasation across the blood-brain barrier (BBB) by 34% (p < 0.05). On analysis stratified by sex, the increase in CMH number was more pronounced in male mice and this correlated with greater creatinine elevation in male compared with female mice. Microglial depletion with PLX3397 diet significantly decreased CMH formation in CKD mice without affecting serum creatinine levels. Incubation of CKD serum significantly reduced transendothelial electrical resistance (TEER) (p < 0.01) and increased sodium fluorescein permeability (p < 0.05) across the endothelial monolayer. Uremic toxins (i.e., indoxyl sulfate, p-cresyl sulfate, and trimethylamine-N-oxide) in combination with urea and lipopolysaccharide induced a marked drop in TEER compared with the control group (p < 0.0001).

Conclusions

CKD promotes the development of CMH in aged mice independent of blood pressure but directly proportional to the degree of renal impairment. These effects of CKD are likely mediated in part by microglia and are associated with BBB impairment. The latter is likely related to gut-derived bacteria-dependent toxins classically associated with CKD. Overall, these findings demonstrate an important role of CKD in the development of cerebral small vessel disease.

Cerebral microhemorrhages (CMH) are the pathological substrate for MRI-demonstrable cerebral microbleeds, which are associated with cognitive impairment and stroke. Aging and hypertension are the main risk factors for CMH. In this study, we investigated the development of CMH in a mouse model of aging and hypertension. Hypertension was induced in aged (17-month-old) female and male C57BL/6J mice via angiotensin II (Ang II), a potent vasoconstrictor. We investigated the vascular origin of CMH using three-dimensional images of 1-mm thick brain sections. We examined Ang II-induced CMH formation with and without telmisartan, an Ang II type 1 receptor (AT1R) blocker. To evaluate the effect of microglia and perivascular macrophages on CMH formation, mice were treated with PLX3397, a selective colony-stimulating factor 1 receptor (CSF1R) inhibitor, to achieve microglial and macrophage depletion. Iba-1 and CD206 labeling were used to study the relative contributions of microglia and macrophages, respectively, on CMH formation. CMH quantification was performed with analysis of histological sections labeled with Prussian blue. Vessels surrounding CMH were primarily of capillary size range (< 10 μm in diameter). Ang II-infused mice exhibited elevated blood pressure (p < 0.0001) and CMH burden (p < 0.001). CMH burden was significantly correlated with mean arterial pressure in mice with and without Ang II (r = 0.52, p < 0.05). Ang II infusion significantly increased Iba-1 immunoreactivity (p < 0.0001), and CMH burden was significantly correlated with Iba-1 in mice with and without Ang II (r = 0.32, p < 0.05). Telmisartan prevented elevation of blood pressure due to Ang II infusion and blocked Ang II-induced CMH formation without affecting Iba-1 immunoreactivity. PLX3397 treatment reduced Iba-1 immunoreactivity in Ang II-infused mice (p < 0.001) and blocked Ang II-induced CMH (p < 0.0001). No significant association between CMH burden and CD206 reactivity was observed. Our findings demonstrate Ang II infusion increases CMH burden. CMH in this model appear to be capillary-derived and Ang II-induced CMH are largely mediated by blood pressure. In addition, microglial activation may represent an alternate pathway for CMH formation. These observations emphasize the continuing importance of blood pressure control and the role of microglia in hemorrhagic cerebral microvascular disease.

Cerebral microhemorrhages (CMH) are the pathological substrate for MRI-demonstrable cerebral microbleeds, which are associated with cognitive impairment and stroke. Aging and hypertension are the main risk factors for CMH. In this study, we investigated the development of CMH in a mouse model of aging and hypertension. Hypertension was induced in aged (17-month-old) female and male C57BL/6J mice via angiotensin II (Ang II), a potent vasoconstrictor. We investigated the vascular origin of CMH using three-dimensional images of 1-mm thick brain sections. We examined Ang II-induced CMH formation with and without telmisartan, an Ang II type 1 receptor (AT1R) blocker. To evaluate the effect of microglia and perivascular macrophages on CMH formation, mice were treated with PLX3397, a selective colony-stimulating factor 1 receptor (CSF1R) inhibitor, to achieve microglial and macrophage depletion. Iba-1 and CD206 labeling were used to study the relative contributions of microglia and macrophages, respectively, on CMH formation. CMH quantification was performed with analysis of histological sections labeled with Prussian blue. Vessels surrounding CMH were primarily of capillary size range (< 10 μm in diameter). Ang II-infused mice exhibited elevated blood pressure (p < 0.0001) and CMH burden (p < 0.001). CMH burden was significantly correlated with mean arterial pressure in mice with and without Ang II (r = 0.52, p < 0.05). Ang II infusion significantly increased Iba-1 immunoreactivity (p < 0.0001), and CMH burden was significantly correlated with Iba-1 in mice with and without Ang II (r = 0.32, p < 0.05). Telmisartan prevented elevation of blood pressure due to Ang II infusion and blocked Ang II-induced CMH formation without affecting Iba-1 immunoreactivity. PLX3397 treatment reduced Iba-1 immunoreactivity in Ang II-infused mice (p < 0.001) and blocked Ang II-induced CMH (p < 0.0001). No significant association between CMH burden and CD206 reactivity was observed. Our findings demonstrate Ang II infusion increases CMH burden. CMH in this model appear to be capillary-derived and Ang II-induced CMH are largely mediated by blood pressure. In addition, microglial activation may represent an alternate pathway for CMH formation. These observations emphasize the continuing importance of blood pressure control and the role of microglia in hemorrhagic cerebral microvascular disease.