Akkermansia muciniphila extracellular vesicles have a protective effect against hypertension.

Akkermansia muciniphila (Am) shows a beneficial role as a probiotic in the treatment of metabolic syndrome. However, the mechanism remains to be elucidated. We tested the hypothesis that Am extracellular vesicles (AmEVs) have a protective effect against hypertension. Extracellular vesicles purified from anaerobically cultured Am were characterized by nanoparticle tracking analysis, transmission electron microscopy, and silver stain after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). AmEVs (1.0 × 1010 log particles/L) or vehicles were added into organ baths to induce vasorelaxation. In addition, AmEVs (1.0 × 108 or 1.0 × 109 particles/kg) or vehicles were injected into the tail veins of Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs) weekly for 4 weeks. Peripheral blood mononuclear cells (PBMCs) and splenocytes isolated from both rat strains were analyzed by flow cytometry, RT-qPCR, and western blot. AmEVs affected neither vascular contraction nor endothelial relaxation in thoracic aortas. Moreover, AmEVs protected against the development of hypertension in SHRs without a serious adverse reaction. Additionally, AmEVs increased the population of T regulatory (Treg) cells and tended to reduce proinflammatory cytokines. These results indicate that AmEVs have a protective effect against hypertension without a serious adverse reaction. Therefore, it is foreseen that AmEVs may be utilized as a novel therapeutic for the treatment of hypertension.

Hypertonic Salt Solution Enhances Inflammatory Responses in Cultured Splenic T-Cells from Dahl Salt-Sensitive Rats but Not Dahl Salt-Resistant Rats.

This study aimed to delineate the effect of sodium chloride on the induction of inflammatory responses and the development of hypertension in Dahl salt-sensitive (SS) and salt-resistant (SR) rats. Splenocytes were isolated from the spleens of SS and SR rats, and cultured on anti-CD3-coated plates for 5 days. The cultured splenic T-cells were challenged with a hypertonic salt solution (0, 20, or 40 mM) in the absence or presence of IL-6 (0, 20, or 60 ng/mL), TGF-ß (0, 5, or 15 ng/mL), or IL-23 (0, 10, or 30 ng/mL), and analyzed via ELISA, flow cytometry, and immunofluorescence. The hypertonic salt solution potentiated IL-17A production, as well as the differentiation of Th17 cells via IL-6/TGF-ß/IL-23, exclusively in SS rats. However, it did not affect IL-10 production or the differentiation of Treg cells in any of the groups. Furthermore, it potentiated the signal of RORγt in IL-6-treated splenic T-cells from SS rats. To summarize, cultured splenic T-cells exhibited enhanced inflammatory responses on exposure to a hypertonic salt solution in SS rats only, which indicated that sodium chloride and inflammatory cytokines synergistically drove the induction of pathogenic Th17 cells and the development of hypertension in this group only.

Anti-Obesity Effect of Chlorin e6-Mediated Photodynamic Therapy on Mice with High-Fat-Diet-Induced Obesity.

This study aimed to evaluate the efficacy of Chlorin e6 (Ce6)-based photodynamic therapy (PDT) for anti-obesity activities in high-fat-diet (HFD)-induced obesity mouse models. We induced obesity in C57BL/6 mice by HFD and administered Ce6 (2.5 or 5 mg/kg) orally with 3 h of incubation. The mice were then exposed to light of high fluence rate (4.96 mW/cm2) or low fluence rate (2.56 mW/cm2) in the designed LED mouse chamber 2-3 days a week for up to 8 weeks. The study also analyzed the pharmacokinetics and optimization of the drug by evaluating the absorption, distribution, metabolism, and excretion (ADME) of Ce6 in the rat models. Both low doses (2.5 mg/kg) and high doses (5 mg/kg) of Ce6 with high irradiation dose showed better anti-obesity effects than other groups with decreased body weight. The lipid accumulation in the liver and adipocyte size in epididymal adipose tissues were found to be decreased by Ce6-PDT in comparison to vehicle-treated HFD groups. We also observed increased levels of the lipidomic biomarkers, such as leptin and LDL cholesterol, while observing decreasing levels of total cholesterol and adiponectin in the Ce6-PDT-treated mice. These findings may provide valuable insight into Ce6-PDT as an alternative and non-invasive therapeutic methodology for obesity and obesity-related diseases.

T helper cell polarity determines salt sensitivity and hypertension development.

High-salt intake is known to induce pathogenic T helper (Th) 17 cells and hypertension, but contrary to what is known, causes hypertension only in salt-sensitive (SS) individuals. Thus, we hypothesized that Th cell polarity determines salt sensitivity and hypertension development. Cultured splenic T cells from Dahl SS and salt-resistant (SR) rats subjected to hypertonic salt solutions were evaluated via ELISA, flow cytometry, immunocytochemistry and RT-qPCR. Seven-week-old SS and SR rats were fed a chow (CD) or high-salt diet (HSD) for 4 weeks, with weekly measurements of systolic blood pressure. The relaxation response of the aorta rings to the cumulative addition of acetylcholine was measured ex vivo. In these experimental animals, the Th cell polarity (Th17 and T regulatory [Treg]), the expression of Th17- or Treg-related genes, and the enrichment of the transcription factors RORγt and FOXP3 on the target gene promoter regions were determined via flow cytometry, RT-qPCR, and chromatin immunoprecipitation. Hypertonic salt solution induced Th17 and Treg cell differentiation in cultured splenic T cells isolated from SS and SR rats, respectively. HSD induced hypertension, endothelial dysfunction and proinflammatory Th17 cell differentiation only in SS rats. The enrichment of RORγt on the promoter regions of Il17a and Il23r increased their expression only in SS rats. Regardless of HSD, SR rats remained normotensive with Treg polarity, causing high Treg-related gene expressions (Il10, Cd25 and Foxp3). This study demonstrated that Th cell polarity determines salt sensitivity and drives hypertension development. SR rats were protected from HSD-associated hypertension via anti-inflammatory Treg polarity.

Single cell transcriptome analyses reveal the roles of B cells in fructose-induced hypertension.

Rationale: While the immune system plays a crucial role in the development of hypertension, the specific contributions of distinct immune cell populations remain incompletely understood. The emergence of single-cell RNA-sequencing (scRNA-seq) technology enables us to analyze the transcriptomes of individual immune cells and to assess the significance of each immune cell type in hypertension development. Objective: We aimed to investigate the hypothesis that B cells play a crucial role in the development of fructose-induced hypertension. Methods and Results: Eight-week-old Dahl salt-sensitive (SS) male rats were divided into two groups and given either tap water (TW) or a 20% fructose solution (HFS) for 4 weeks. Systolic blood pressure was measured using the tail-cuff method. ScRNA-seq analysis was performed on lamina propria cells (LPs) and peripheral blood mononuclear cells (PBMCs) obtained from SS rats subjected to either TW or HFS. The HFS treatment induced hypertension in the SS rats. The analysis revealed 27 clusters in LPs and 28 clusters in PBMCs, allowing for the identification and characterization of various immune cell types within each cluster. Specifically, B cells and follicular helper T (Tfh) cells were prominent in LPs, while B cells and M1 macrophages dominated PBMCs in the HFS group. Moreover, the HFS treatment triggered an increase in the number of B cells in both LPs and PBMCs, accompanied by activation of the interferon pathway. Conclusions: The significant involvement of B cells in intestinal and PBMC responses indicates their pivotal contribution to the development of hypertension. This finding suggests that targeting B cells could be a potential strategy to mitigate high blood pressure in fructose-induced hypertension. Moreover, the simultaneous increase in follicular B cells and Tfh cells in LPs, along with the upregulation of interferon pathway genes in B cells, underscores a potential autoimmune factor contributing to the pathogenesis of fructose-induced hypertension in the intestine.

Dahl salt-resistant rats are protected against angiotensin II-induced hypertension.

Angiotensin II is a potent endogenous vasoconstrictor that induces oxidative stress in hypertensive rodent models. Dahl salt-resistant (SR) rats are protected against hypertension after high salt or high fructose intake. However, whether these rats are also protected against angiotensin II-mediated hypertension has not been investigated. Dahl salt-sensitive (SS) and SR rats were infused with angiotensin II (10 or 50 ng/kg/min) or vehicle via a mini-osmotic pump for 2 weeks. Blood pressure was measured using the tail-cuff method. Paraffin sections of the thoracic aortas and kidneys were stained using hematoxylin/eosin or Masson trichrome. Renal gene expression was measured using reverse transcription-quantitative polymerase chain reaction. Angiotensin II (50 ng/kg/min) induced hypertension in SS rats, but not in SR rats, although low doses of angiotensin II (10 ng/kg/min) transiently increased blood pressure in SS rats. Angiotensin II (50 ng/kg/min) did not induce morphological changes in the aortic walls or kidneys. Angiotensin II (50 ng/kg/min) induced the expression of At1rb, Nox2, Il-17ra, Il-23r, Tgf-ß, Il-1ß and Il-6 in SS rats, but not in SR rats. In conclusion, SR rats were protected against angiotensin II-induced hypertension. This result implies that the genetic trait that determines salt sensitivity may also determine susceptibility to hypertension in response to vasoconstrictors.

Transfer of Th17 from Adult Spontaneous Hypertensive Rats Accelerates Development of Hypertension in Juvenile Spontaneous Hypertensive Rats.

Hypertension develops in the recipient rats that are transferred with the activated T helper (Th) 17 cells of the donor rats exposed to high-fructose or high-salt intake. This result suggests that a pathologic Th17 cell plays a role in the development and maintenance of hypertension. Here, we tested the hypothesis that the transfer of Th17 cells from adult spontaneous hypertensive rats (SHR) accelerates the development of hypertension in juvenile SHR. The tail-cuff method was used to measure systolic blood pressure. T cell (Th17 and regulatory T (Treg)) profiling was analyzed by flow cytometry. The expressions of Th17-related interleukin- (IL-) 17A and Treg-related IL-10 were measured by ELISA. Th17 cells isolated from adult SHR were intraperitoneally injected into juvenile recipient SHR and Wistar-Kyoto rats (WKY). SHR exhibited prominent development of hypertension at 15 weeks. The proportion of CD4+IL-17A+ (Th17) cells among Th cells increased whereas the proportion of CD4+FoxP3+ (Treg) cells decreased in SHR, as compared to WKY. The serum levels of IL-17A increased gradually with aging in SHR, but the serum levels of IL-10 did not. The serum levels of IL-17A and IL-10 seemed to be well related to the proportion of Th17 cells and Treg cells, respectively. Injection of Th17 cells isolated from adult SHR accelerates the development of hypertension in juvenile SHR but not in juvenile WKY though it increased the proportion of Th17 cells in juvenile recipient WKY and SHR. The transfer of Th17 cells from adult SHR accelerates the development of hypertension in juvenile SHR. These results implicate that the hypertension in SHR is ascribed to activation of Th17 cells.

Enhanced delivery of liposomes to lung tumor through targeting interleukin-4 receptor on both tumor cells and tumor endothelial cells.

A growing body of evidence suggests that pathological lesions express tissue-specific molecular targets or biomarkers within the tissue. Interleukin-4 receptor (IL-4R) is overexpressed in many types of cancer cells, including lung cancer. Here we investigated the properties of IL-4R-binding peptide-1 (IL4RPep-1), a CRKRLDRNC peptide, and its ability to target the delivery of liposomes to lung tumor. IL4RPep-1 preferentially bound to H226 lung tumor cells which express higher levers of IL-4R compared to H460 lung tumor cells which express less IL-4R. Mutational analysis revealed that C1, R2, and R4 residues of IL4RPep-1 were the key binding determinants. IL4RPep-1-labeled liposomes containing doxorubicin were more efficiently internalized in H226 cells and effectively delivered doxorubicin into the cells compared to unlabeled liposomes. In vivo fluorescence imaging of nude mice subcutaneously xenotransplanted with H226 tumor cells indicated that IL4RPep-1-labeled liposomes accumulate more efficiently in the tumor and inhibit tumor growth more effectively compared to unlabeled liposomes. Interestingly, expression of IL-4R was high in vascular endothelial cells of tumor, while little was detected in vascular endothelial cells of control organs including the liver. IL-4R expression in cultured human vascular endothelial cells was also up-regulated when activated by a pro-inflammatory cytokine tumor necrosis factor-α. Moreover, the up-regulation of IL-4R expression was observed in primary human lung cancer tissues. These results indicate that IL-4R-targeting nanocarriers may be a useful strategy to enhance drug delivery through the recognition of IL-4R in both tumor cells and tumor endothelial cells.