Targeting cytokine-like protein FAM3D lowers blood pressure in hypertension.

Current antihypertensive options still incompletely control blood pressure, suggesting the existence of uncovered pathogenic mechanisms. Here, whether cytokine-like protein family with sequence similarity 3, member D (FAM3D) is involved in hypertension etiology is evaluated. A case-control study exhibits that FAM3D is elevated in patients with hypertension, with a positive association with odds of hypertension. FAM3D deficiency significantly ameliorates angiotensin II (AngII)-induced hypertension in mice. Mechanistically, FAM3D directly causes endothelial nitric oxide synthase (eNOS) uncoupling and impairs endothelium-dependent vasorelaxation, whereas 2,4-diamino-6-hydroxypyrimidine to induce eNOS uncoupling abolishes the protective effect of FAM3D deficiency against AngII-induced hypertension. Furthermore, antagonism of formyl peptide receptor 1 (FPR1) and FPR2 or the suppression of oxidative stress blunts FAM3D-induced eNOS uncoupling. Translationally, targeting endothelial FAM3D by adeno-associated virus or intraperitoneal injection of FAM3D-neutralizing antibodies markedly ameliorates AngII- or deoxycorticosterone acetate (DOCA)-salt-induced hypertension. Conclusively, FAM3D causes eNOS uncoupling through FPR1- and FPR2-mediated oxidative stress, thereby exacerbating the development of hypertension. FAM3D may be a potential therapeutic target for hypertension.

Urate-Lowering Therapy Inhibits Thoracic Aortic Aneurysm and Dissection Formation in Mice.

BACKGROUND: Thoracic aortic aneurysm and dissection (TAAD) is a highly lethal vascular disease without effective drug therapy. Whether elevated serum concentrations of uric acid are involved in TAAD development remains unclear. METHODS: Serum uric acid levels were detected in different TAAD mouse models and patients. The urate-lowering drug allopurinol was administered in the drinking water of TAAD mice. Adenine diet-induced mice were established to investigate the role of hyperuricemia in TAAD formation and RNA-sequencing of thoracic aortas from these mice was performed. RESULTS: We found serum uric acid levels were elevated in various mouse TAAD models, including mice fed a ß-aminopropionitrile diet, Marfan mice with fibrillin-1 haploinsufficiency (Fbn1C1041G/+), and ApoE-/- mice infused with Ang II (angiotensin II), as well as in patients with TAAD. Administration of urate-lowering drug allopurinol in the drinking water significantly alleviated TAAD formation in ß-aminopropionitrile-treated mice, Fbn1C1041G/+ mice, and Ang II-infused ApoE-/- mice. Moreover, an adenine diet was used to induce hyperuricemia in mice. Intriguingly, a 4-week adenine diet feeding directly induced TAAD formation characterized by increased maximal thoracic aortic diameters and severe elastin degradation, which were ameliorated by allopurinol. Unbiased RNA-sequencing in mouse thoracic aortas suggested that FcγR (Fc gamma receptor) was upregulated upon adenine diet, but reciprocally repressed by allopurinol. Mechanistically, hyperuricemia activated FcγR-mediated ERK1/2 (extracellular signal-regulated kinase 1/2) phosphorylation to induce macrophage inflammation and TAAD development, which was abrogated by allopurinol or FcγR deficiency. CONCLUSIONS: This study uncovered an important and previously unrecognized role of hyperuricemia in mediating the pathogenesis of TAAD, and uric acid-lowering drug may represent a promising therapeutic approach for TAAD.

Activin Receptor-Like Kinase 3 Directly Couples Gαq (Guanine Nucleotide-Binding Protein Subunit αq)/ Gαq (Guanine Nucleotide-Binding Protein Subunit α11) to Regulate Vascular Contractility.

BACKGROUND: Vascular smooth muscle cell (VSMC) contractility is critical for blood pressure regulation and vascular homeostasis. Identifying the key molecule that maintains VSMC contractility may provide a novel therapeutic target for vascular remodeling. ALK3 (activin receptor-like kinase 3) is a serine/threonine kinase receptor, and deletion of ALK3 causes embryonic lethality. However, little is known about the role of ALK3 in postnatal arterial function and homeostasis. METHODS: We conducted in vivo studies in a tamoxifen-induced postnatal VSMC-specific ALK3 deletion mice suitable for analysis of blood pressure and vascular contractility. Additionally, the role of ALK3 on VSMC was determined using Western blot, collagen-based contraction assay and traction force microscopy. Furthermore, interactome analysis were performed to identify the ALK3-associated proteins and bioluminescence resonance energy transfer assay was used to characterize Gαq activation. RESULTS: ALK3 deficiency in VSMC led to spontaneous hypotension and impaired response to angiotensin II in mice. In vivo and in vitro data revealed that ALK3 deficiency impaired contraction force generation by VSMCs, repressed the expression of contractile proteins, and inhibited the phosphorylation of myosin light chain. Mechanistically, Smad1/5/8 signaling mediated the ALK3-modulated contractile protein expressions but not myosin light chain phosphorylation. Furthermore, interactome analysis revealed that ALK3 directly interacted with and activated Gαq (guanine nucleotide-binding protein subunit αq)/Gα11 (guanine nucleotide-binding protein subunit α11), thereby stimulating myosin light chain phosphorylation and VSMC contraction. CONCLUSIONS: Our study revealed that in addition to canonical Smad1/5/8 signaling, ALK3 modulates VSMC contractility through direct interaction with Gαq/Gα11, and therefore, might serve as a potential target for modulating aortic wall homeostasis.

MicroRNA-Disease Network Analysis Repurposes Methotrexate for the Treatment of Abdominal Aortic Aneurysm in Mice.

Abdominal aortic aneurysm (AAA) is a permanent dilatation of the abdominal aorta and is highly lethal. The main purpose of the current study is to search for noninvasive medical therapies for AAA, for which there is currently no effective drug therapy. Network medicine represents a cutting-edge technology, as analysis and modeling of disease networks can provide critical clues regarding the etiology of specific diseases and which therapeutics may be effective. Here, we proposed a novel algorithm to quantify disease relations based on a large accumulated microRNA-disease association dataset and then built a disease network covering 15 disease classes and 304 diseases. Analysis revealed some patterns for these diseases. For instance, diseases tended to be clustered and coherent in the network. Surprisingly, we found that AAA showed the strongest similarity with rheumatoid arthritis and systemic lupus erythematosus, both of which are autoimmune diseases, suggesting that AAA could be one type of autoimmune disease in etiology. Based on this observation, we further hypothesized that drugs for autoimmune disease could be repurposed for the prevention and therapy of AAA. Finally, animal experiments confirmed that methotrexate, a drug for autoimmune disease, was able to alleviated the formation and development of AAA.

Nutrient regulation of biological nitrogen fixation across the tropical western North Pacific.

Nitrogen fixation is critical for the biological productivity of the ocean, but clear mechanistic controls on this process remain elusive. Here, we investigate the abundance, activity, and drivers of nitrogen-fixing diazotrophs across the tropical western North Pacific. We find a basin-scale coherence of diazotroph abundances and N2 fixation rates with the supply ratio of iron:nitrogen to the upper ocean. Across a threshold of increasing supply ratios, the abundance of nifH genes and N2 fixation rates increased, phosphate concentrations decreased, and bioassay experiments demonstrated evidence for N2 fixation switching from iron to phosphate limitation. In the northern South China Sea, supply ratios were hypothesized to fall around this critical threshold and bioassay experiments suggested colimitation by both iron and phosphate. Our results provide evidence for iron:nitrogen supply ratios being the most important factor in regulating the distribution of N2 fixation across the tropical ocean.

Unspliced XBP1 Counteracts ß-Catenin to Inhibit Vascular Calcification.

BACKGROUND: Vascular calcification is a prevalent complication in chronic kidney disease and contributes to increased cardiovascular morbidity and mortality. XBP1 (X-box binding protein 1), existing as the XBP1u (unspliced XBP1) and XBP1s (spliced XBP1) forms, is a key component of the endoplasmic reticulum stress involved in vascular diseases. However, whether XBP1u participates in the development of vascular calcification remains unclear. METHODS: We aim to investigate the role of XBP1u in vascular calcification. XBP1u protein levels were reduced in high phosphate-induced calcified vascular smooth muscle cells, calcified aortas from mice with adenine diet-induced chronic renal failure, and calcified radial arteries from patients with chronic renal failure. RESULTS: Inhibition of XBP1u rather than XBP1s upregulated in the expression of the osteogenic markers Runx2 (runt-related transcription factor 2) and Msx2 (msh homeobox 2), and exacerbated high phosphate-induced vascular smooth muscle cell calcification, as verified by calcium deposition and Alizarin red S staining. In contrast, XBP1u overexpression in high phosphate-induced vascular smooth muscle cells significantly inhibited osteogenic differentiation and calcification. Consistently, smooth muscle cell-specific XBP1 deficiency in mice markedly aggravated the adenine diet- and 5/6 nephrectomy-induced vascular calcification compared with that in the control littermates. Further interactome analysis revealed that XBP1u is bound directly to ß-catenin, a key regulator of vascular calcification, via amino acid (aa) 205-230 in its C-terminal degradation domain. XBP1u interacted with ß-catenin to promote its ubiquitin-proteasomal degradation and thus inhibited ß-catenin/TCF (T-cell factor)-mediated Runx2 and Msx2 transcription. Knockdown of ß-catenin abolished the effect of XBP1u deficiency on vascular smooth muscle cell calcification, suggesting a ß-catenin-mediated mechanism. Moreover, the degradation of ß-catenin promoted by XBP1u was independent of GSK-3ß (glycogen synthase kinase 3ß)-involved destruction complex. CONCLUSIONS: Our study identified XBP1u as a novel endogenous inhibitor of vascular calcification by counteracting ß-catenin and promoting its ubiquitin-proteasomal degradation, which represents a new regulatory pathway of ß-catenin and a promising target for vascular calcification treatment.

Rapamycin prevents thoracic aortic aneurysm and dissection in mice.

OBJECTIVE: The purpose of this study was to investigate whether rapamycin inhibits the development of thoracic aortic aneurysm and dissection (TAAD) in mice. METHODS: Three-week-old C57BL/6J male mice were fed a normal diet and randomized into a control group (n = 6), ß-aminopropionitrile fumarate (BAPN) group (Gp A; n = 15), BAPN plus rapamycin (5 mg) group (Gp B; n = 8), and BAPN plus rapamycin (10 mg) group (Gp C; n = 8). Gp A, Gp B, and Gp C were administered BAPN (1 g/kg/d) for 4 weeks. One week after BAPN administration, Gp B and Gp C were treated with rapamycin (5 mg/kg/d or 10 mg/kg/d) through gavage for 21 days. Thoracic aortas were harvested for Western blot and immunofluorescence staining at day 14 and for morphologic and histologic analyses at day 28. RESULTS: BAPN treatment induced TAAD formation in mice. The incidence of TAAD in control, Gp A, Gp B, and Gp C mice was 0%, 80%, 25%, and 37.5%, respectively. Smaller thoracic aortic diameters (ascending aorta and arch) were observed in Gp B and Gp C mice than in Gp A mice (Gp B vs Gp A: ascending aorta, ex vivo, 1.07 ± 0.21 mm vs 1.80 ± 0.67 mm [P < .05]; aortic arch, ex vivo, 1.51 ± 0.40 mm vs 2.70 ± 1.06 mm [P < .05]; Gp C vs Gp A: ascending aortas, ex vivo, 1.10 ± 0.33 mm vs 1.80 ± 0.67 mm [P < .05]; aortic arch, ex vivo, 1.55 ± 0.56 mm vs 2.70 ± 1.06 mm [P < .05]). TAAD mice exhibited elastin fragmentation, abundant inflammatory cell infiltration, and significantly increased matrix metalloproteinase production in the aorta, and rapamycin treatment alleviated these changes. The protein levels of p-S6K and p-S6 in TAAD aortic tissues increased significantly, whereas they were suppressed by rapamycin. CONCLUSIONS: Rapamycin suppressed TAAD formation, probably by inhibition of mechanistic target of rapamycin signaling and reduction of inflammatory cell infiltration and matrix metalloproteinase 9 production. Targeting of the mechanistic target of rapamycin signaling pathway using rapamycin may be a favorable modulation for the clinical treatment of TAAD.

Probing active microbes involved in Bt-containing rice straw decomposition.

Transgenic Bacillus thuringiensis (Bt) rice extends significant protection against insect pests and meets the increasing demands for food and energy. Many studies have been conducted investigating the impacts of Bt rice to the agricultural ecosystem, but much less attention has been given to efforts attempting to determine how the presence of Bt rice influences and shapes the microbial community, especially the active microbes. Stable isotope probing and high-throughput sequencing were employed to explore the active microbes involved in Bt-containing straw decomposition. Compared to its near isoline, the Bt straw contained higher contents of total N, total P, total K, lignin, cellulose, and Cry1Ab toxin protein. These chemical differences did not affect the decomposition rate but significantly changed the active microbial decomposer communities. During the decomposition of Bt-containing straw, fungi were more affected than bacteria. Agromyces, Terrabacter, Microbacterium, Glycomyces, and Kribbella were the most representative unique (existed only in the Bt treatments and appeared at the early stage) bacterial genera, and Trichoderma was the most representative unique fungal genus in the Bt straw decomposition. By using similarity index calculation and function prediction, the significant differences between Bt straw and non-Bt straw treatments were found to be transient for both microbial taxa and functional traits. These results suggested that Bt rice has a significant but transient impact on soil microbes in terms of microbial straw decomposition.

Cartilage oligomeric matrix protein is a novel notch ligand driving embryonic stem cell differentiation towards the smooth muscle lineage.

Cartilage oligomeric matrix protein (COMP), a protective component of vascular extracellular matrix (ECM), maintains the homeostasis of mature vascular smooth muscle cells (VSMCs). However, whether COMP modulates the differentiation of stem cells towards the smooth muscle lineage is still elusive. Firstly, purified mouse COMP directly induced mouse embryonic stem cell (ESC) differentiation into VSMCs both in vitro and in vivo, while the silencing of endogenous COMP markedly inhibited ESC-VSMC differentiation. RNA-Sequencing revealed that Notch signaling was significantly activated by COMP during ESC-VSMC differentiation, whereas the inhibition of Notch signaling attenuated COMP-directed ESC-VSMC differentiation. Furthermore, COMP deficiency inhibited Notch activation and VSMC differentiation in mice. Through silencing distinct Notch receptors, we identified that Notch1 mainly mediated COMP-initiated ESC-VSMC differentiation. Mechanistically, COMP N-terminus directly interacted with the EGF11-12 domain of Notch1 and activated Notch1 signaling, as evidenced by co-immunoprecipitation and mammalian two-hybrid assay. In conclusion, COMP served as a potential ligand of Notch1, thereby driving ESC-VSMC differentiation.

Unspliced XBP1 Confers VSMC Homeostasis and Prevents Aortic Aneurysm Formation via FoxO4 Interaction.

RATIONALE: Although not fully understood, the phenotypic transition of vascular smooth muscle cells exhibits at the early onset of the pathology of aortic aneurysms. Exploring the key regulators that are responsible for maintaining the contractile phenotype of vascular smooth muscle cells (VSMCs) may confer vascular homeostasis and prevent aneurysmal disease. XBP1 (X-box binding protein 1), which exists in a transcriptionally inactive unspliced form (XBP1u) and a spliced active form (XBP1s), is a key component in response to endoplasmic reticular stress. Compared with XBP1s, little is known about the role of XBP1u in vascular homeostasis and disease. OBJECTIVE: We aim to investigate the role of XBP1u in VSMC phenotypic switching and the pathogenesis of aortic aneurysms. METHODS AND RESULTS: XBP1u, but not XBP1s, was markedly repressed in the aorta during the early onset of aortic aneurysm in both angiotensin II-infused apolipoprotein E knockout (ApoE-/-) and CaPO4 (calcium phosphate)-induced C57BL/6J murine models, in parallel with a decrease in smooth muscle cell contractile apparatus proteins. In vivo studies revealed that XBP1 deficiency in smooth muscle cells caused VSMC dedifferentiation, enhanced vascular inflammation and proteolytic activity, and significantly aggravated both thoracic and abdominal aortic aneurysms in mice. XBP1 deficiency, but not an inhibition of XBP1 splicing, induced VSMC switching from the contractile phenotype to a proinflammatory and proteolytic phenotype. Mechanically, in the cytoplasm, XBP1u directly associated with the N terminus of FoxO4 (Forkhead box protein O 4), a recognized repressor of VSMC differentiation via the interaction and inhibition of myocardin. Blocking the XBP1u-FoxO4 interaction facilitated nuclear translocation of FoxO4, repressed smooth muscle cell marker genes expression, promoted proinflammatory and proteolytic phenotypic transitioning in vitro, and stimulated aortic aneurysm formation in vivo. CONCLUSIONS: Our study revealed the pivotal role of the XBP1u-FoxO4-myocardin axis in maintaining the VSMC contractile phenotype and providing protection from aortic aneurysm formation.