Genetic fusion of CCL11 to antigens enhances antigenicity in nucleic acid vaccines and eradicates tumor mass through optimizing T-cell response.

Nucleic acid vaccines have shown promising potency and efficacy for cancer treatment with robust and specific T-cell responses. Improving the immunogenicity of delivered antigens helps to extend therapeutic efficacy and reduce dose-dependent toxicity. Here, we systematically evaluated chemokine-fused HPV16 E6/E7 antigen to improve the cellular and humoral immune responses induced by nucleotide vaccines in vivo. We found that fusion with different chemokines shifted the nature of the immune response against the antigens. Although a number of chemokines were able to amplify specific CD8 + T-cell or humoral response alone or simultaneously. CCL11 was identified as the most potent chemokine in improving immunogenicity, promoting specific CD8 + T-cell stemness and generating tumor rejection. Fusing CCL11 with E6/E7 antigen as a therapeutic DNA vaccine significantly improved treatment effectiveness and caused eradication of established large tumors in 92% tumor-bearing mice (n = 25). Fusion antigens with CCL11 expanded the TCR diversity of specific T cells and induced the infiltration of activated specific T cells, neutrophils, macrophages and dendritic cells (DCs) into the tumor, which created a comprehensive immune microenvironment lethal to tumor. Combination of the DNA vaccine with anti-CTLA4 treatment further enhanced the therapeutic effect. In addition, CCL11 could also be used for mRNA vaccine design. To summarize, CCL11 might be a potent T cell enhancer against cancer.

Inhibition of miR-101-3p prevents human aortic valve interstitial cell calcification through regulation of CDH11/SOX9 expression.

BACKGROUND: Calcific aortic valve disease (CAVD) is the second leading cause of adult heart diseases. The purpose of this study is to investigate whether miR-101-3p plays a role in the human aortic valve interstitial cells (HAVICs) calcification and the underlying mechanisms. METHODS: Small RNA deep sequencing and qPCR analysis were used to determine changes in microRNA expression in calcified human aortic valves. RESULTS: The data showed that miR-101-3p levels were increased in the calcified human aortic valves. Using cultured primary HAVICs, we demonstrated that the miR-101-3p mimic promoted calcification and upregulated the osteogenesis pathway, while anti-miR-101-3p inhibited osteogenic differentiation and prevented calcification in HAVICs treated with the osteogenic conditioned medium. Mechanistically, miR-101-3p directly targeted cadherin-11 (CDH11) and Sry-related high-mobility-group box 9 (SOX9), key factors in the regulation of chondrogenesis and osteogenesis. Both CDH11 and SOX9 expressions were downregulated in the calcified human HAVICs. Inhibition of miR-101-3p restored expression of CDH11, SOX9 and ASPN and prevented osteogenesis in HAVICs under the calcific condition. CONCLUSION: miR-101-3p plays an important role in HAVIC calcification through regulation of CDH11/SOX9 expression. The finding is important as it reveals that miR-1013p may be a potential therapeutic target for calcific aortic valve disease.

Klotho Deficiency Causes Heart Aging via Impairing the Nrf2-GR Pathway.

RATIONALE: Cardiac aging is an important contributing factor for heart failure, which affects a large population but remains poorly understood. OBJECTIVE: The purpose of this study is to investigate whether Klotho plays a role in cardiac aging. METHODS AND RESULTS: Heart function declined in old mice (24 months), as evidenced by decreases in fractional shortening, ejection fraction, and cardiac output. Heart size and weight, cardiomyocyte size, and cardiac fibrosis were increased in old mice, indicating that aging causes cardiac hypertrophy and remodeling. Circulating Klotho levels were dramatically decreased in old mice, which prompted us to investigate whether the Klotho decline may cause heart aging. We found that Klotho gene mutation (KL-/-) largely decreased serum klotho levels and impaired heart function. Interestingly, supplement of exogenous secreted Klotho prevented heart failure, hypertrophy, and remodeling in both old mice and KL (-/-) mice. Secreted Klotho treatment inhibited excessive cardiac oxidative stress, senescence and apoptosis in old mice and KL (-/-) mice. Serum phosphate levels in KL (-/-) mice were kept in the normal range, suggesting that Klotho deficiency-induced heart aging is independent of phosphate metabolism. Mechanistically, Klotho deficiency suppressed GR (glutathione reductase) expression and activity in the heart via inhibition of transcription factor Nrf2 (nuclear factor-erythroid 2 p45-related factor 2). Furthermore, cardiac-specific overexpression of GR prevented excessive oxidative stress, apoptosis, and heart failure in both old and KL (-/-) mice. CONCLUSIONS: Klotho deficiency causes cardiac aging via impairing the Nrf2-GR pathway. Supplement of exogenous secreted Klotho represents a promising therapeutic strategy for aging-associated cardiomyopathy and heart failure.

Aging impairs arterial compliance via Klotho-mediated downregulation of B-cell population and IgG levels.

OBJECTIVE: Aging is associated with compromised immune function and arterial remodeling and stiffness. The purpose of this study is to investigate whether in vivo AAV-based delivery of secreted Klotho (SKL) gene (AAV-SKL) improves aging- and senescence-associated immune dysfunction and arterial stiffness. METHODS AND RESULTS: Senescence-accelerated mice prone strain 1 (SAMP1, 10 months) and old mice (20 months) were used. Serum SKL levels, B-cell population and serum IgG levels were markedly decreased in SAMP1 and old mice. Rescue of downregulation of serum SKL levels by in vivo AAV2-based delivery of SKL gene (AAV-SKL) increased B-cell population and serum IgG levels and attenuated arterial stiffness in SAMP1 and old mice. Thus, Klotho deficiency may play a role in senescence- and aging-associated humoral immune dysfunction and arterial stiffness. Vascular infiltration of inflammatory cells and expression of TGFß1, collagen 1, scleraxis, MMP-2 and MMP-9 were increased while the elastin level was decreased in aortas of SAMP1 and old mice which can be rescued by AAV-SKL. Interestingly, treatment with IgG effectively rescued arterial inflammation and remodeling and attenuated arterial stiffness and hypertension in aging mice. In cultured B-lymphoblast cells, we further showed that SKL regulates B-cell proliferation and maturation partly via the NFkB pathway. CONCLUSION: Aging-associated arterial stiffening may be largely attributed to downregulation of B-cell population and serum IgG levels. AAV-SKL attenuates arterial stiffness in aging mice partly via restoring B-cell population and serum IgG levels which attenuates aging-associated vascular inflammation and arterial remodeling.

Klotho deficiency-induced arterial calcification involves osteoblastic transition of VSMCs and activation of BMP signaling.

Klotho is an aging-suppressor gene. The purpose of this study was to investigate whether Klotho deficiency affects arterial structure. We found that Klotho-deficient (kl/kl) mice developed severe arterial calcification and elastin fragmentation. Klotho-deficient mice demonstrated higher levels of bone morphogenetic proteins (BMP2, BMP4) and runt-related transcription factor 2 (RUNX2) in aortas, indicating that Klotho deficiency upregulates expression of BMP2 and RUNX2 (a key transcription factor in osteoblasts). To exclude the potential involvement of hyperphosphatemia in arterial calcification, Klotho-deficient mice were given a low phosphate diet (0.2%). The low phosphate diet normalized blood phosphate levels and abolished calcification in the lungs and kidneys, but it did not prevent calcification in the aortas in Klotho-deficient mice. Thus, Klotho deficiency per se might play a causal role in the pathogenesis of arterial calcification, which is independent of hyperphosphatemia. In cultured mouse aortic smooth muscle cells (ASMCs), Klotho-deficient serum-induced transition of ASMCs to osteoblasts. Klotho-deficient serum promoted BMP2/vitamin D3-induced protein expression of PIT2 and RUNX2, phosphorylation of SMAD1/5/8 and SMAD2/3, and extracellular matrix calcification. Interestingly, treatments with recombinant Klotho protein abolished BMP2/vitamin D3-induced osteoblastic transition and morphogenesis and calcification. Therefore, Klotho is a critical regulator in the maintenance of normal arterial homeostasis. Klotho deficiency-induced arterial calcification is an active process that involves the osteoblastic transition of SMCs and activation of the BMP2-RUNX2 signaling.

The Antiaging Gene Klotho Regulates Proliferation and Differentiation of Adipose-Derived Stem Cells.

Klotho was originally discovered as an aging-suppressor gene. The purpose of this study was to investigate whether secreted Klotho (SKL) affects the proliferation and differentiation of adipose-derived stem cells (ADSCs). RT-PCR and Western blot analysis showed that short-form Klotho was expressed in mouse ADSCs. The Klotho gene mutation KL(-/-) significantly decreased proliferation of ADSCs and expression of pluripotent transcription factors (Nanog, Sox-2, and Oct-4) in mice. The adipogenic differentiation of ADSCs was also decreased in KL(-/-) mice. Incubation with Klotho-deficient medium decreased ADSC proliferation, pluripotent transcription factor levels, and adipogenic differentiation, which is similar to what was found in KL(-/-) mice. These results indicate that Klotho deficiency suppresses ADSC proliferation and differentiation. Interestingly, treatment with recombinant SKL protein rescued the Klotho deficiency-induced impairment in ADSC proliferation and adipogenic differentiation. SKL also regulated ADSCs' differentiation to other cell lineages (osteoblasts, myofibroblasts), indicating that SKL maintains stemness of ADSCs. It is intriguing that overexpression of SKL significantly increased PPAR-γ expression and lipid formation in ADSCs following adipogenic induction, indicating enhanced adipogenic differentiation. Overexpression of SKL inhibited expression of TGFß1 and its downstream signaling mediator Smad2/3. This study demonstrates, for the first time, that SKL is essential to the maintenance of normal proliferation and differentiation in ADSCs. Klotho regulates adipogenic differentiation in ADSCs, likely via inhibition of TGFß1 and activation of PPAR-γ. Stem Cells 2016;34:1615-1625.

Antiaging Gene Klotho Regulates Adrenal CYP11B2 Expression and Aldosterone Synthesis.

Deficiency of the antiaging gene Klotho (KL) induces renal damage and hypertension through unknown mechanisms. In this study, we assessed whether KL regulates expression of CYP11B2, a key rate-limiting enzyme in aldosterone synthesis, in adrenal glands. We found that haplodeficiency of KL(+/-) in mice increased the plasma level of aldosterone by 16 weeks of age, which coincided with spontaneous and persistent elevation of BP. Blockade of aldosterone actions by eplerenone reversed KL deficiency-induced hypertension and attenuated the kidney damage. Protein expression of CYP11B2 was upregulated in adrenal cortex of KL(+/-) mice. KL and CYP11B2 proteins colocalized in adrenal zona glomerulosa cells. Silencing of KL upregulated and overexpression of KL downregulated CYP11B2 expression in human adrenocortical cells. Notably, silencing of KL decreased expression of SF-1, a negative transcription factor of CYP11B2, but increased phosphorylation of ATF2, a positive transcription factor of CYP11B2, which may contribute to upregulation of CYP11B2 expression. Therefore, these results show that KL regulates adrenal CYP11B2 expression. KL deficiency-induced spontaneous hypertension and kidney damage may be partially attributed to the upregulation of CYP11B2 expression and aldosterone synthesis.

Klotho gene deficiency causes salt-sensitive hypertension via monocyte chemotactic protein-1/CC chemokine receptor 2-mediated inflammation.

Klotho (KL) is a newly discovered aging suppressor gene. In mice, the KL gene extends the lifespan when overexpressed and shortens the lifespan when disrupted. This study investigated if KL deficiency affects BP and salt sensitivity using KL mutant heterozygous (+/-) mice and wild-type (WT) mice (9 weeks of age, 16 mice per group). Notably, systolic BP in KL(+/-) mice began to increase at the age of 15 weeks, reached a peak level at the age of 17 weeks, and remained elevated thereafter, whereas systolic BP remained consistent in WT mice. High salt (HS) intake further increased BP in KL(+/-) mice but did not affect BP in WT mice. Blockade of CC chemokine receptor 2 (CCR2), involved in monocyte chemotaxis, by a specific CCR2 antagonist (INCB3284) abolished the HS-induced increase in BP in KL(+/-) mice. Furthermore, HS loading substantially increased the expression of monocyte chemotactic protein-1 and the infiltration of macrophages and T cells in kidneys in KL(+/-) mice, and treatment with INCB3284 abolished these effects. Treatment of KL(+/-) mice with INCB3284 also attenuated the increased renal expressions of serum glucocorticoid-regulated kinase 1, thiazide-sensitive NaCl cotransporter, and ATP synthase ß along with the renal structural damage and functional impairment induced by HS loading. In conclusion, KL deficiency caused salt-sensitive hypertension and renal damage by CCR2-mediated inflammation.

Molecular mechanisms of pulmonary arterial remodeling.

Pulmonary arterial hypertension (PAH) is characterized by a persistent elevation of pulmonary arterial pressure and pulmonary arterial remodeling with unknown etiology. Current therapeutics available for PAH are primarily directed at reducing the pulmonary blood pressure through their effects on the endothelium. It is well accepted that pulmonary arterial remodeling is primarily due to excessive pulmonary arterial smooth muscle cell (PASMC) proliferation that leads to narrowing or occlusion of the pulmonary vessels. Future effective therapeutics will be successful in reversing the vascular remodeling in the pulmonary arteries and arterioles. The purpose of this review is to provide updated information on molecular mechanisms involved in pulmonary arterial remodeling with a focus on growth factors, transcription factors, and epigenetic pathways in PASMC proliferation. In addition, this review will highlight novel therapeutic strategies for potentially reversing PASMC proliferation.

TNFα Induces DNA and Histone Hypomethylation and Pulmonary Artery Smooth Muscle Cell Proliferation Partly via Excessive Superoxide Formation.

Objective: The level of tumor necrosis factor-α (TNF-α) is upregulated during the development of pulmonary vascular remodeling and pulmonary hypertension. A hallmark of pulmonary arterial (PA) remodeling is the excessive proliferation of PA smooth muscle cells (PASMCs). The purpose of this study is to investigate whether TNF-α induces PASMC proliferation and explore the potential mechanisms. Methods: PASMCs were isolated from 8-week-old male Sprague-Dawley rats and treated with 0, 20, or 200 ng/mL TNF-α for 24 or 48 h. After treatment, cell number, superoxide production, histone acetylation, DNA methylation, and histone methylation were assessed. Results: TNF-α treatment increased NADPH oxidase activity, superoxide production, and cell numbers compared to untreated controls. TNF-α-induced PASMC proliferation was rescued by a superoxide dismutase mimetic tempol. TNF-α treatment did not affect histone acetylation at either dose but did significantly decrease DNA methylation. DNA methyltransferase 1 activity was unchanged by TNF-α treatment. Further investigation using QRT-RT-PCR revealed that GADD45-α, a potential mediator of DNA demethylation, was increased after TNF-α treatment. RNAi inhibition of GADD45-α alone increased DNA methylation. TNF-α impaired the epigenetic mechanism leading to DNA hypomethylation, which can be abolished by a superoxide scavenger tempol. TNF-α treatment also decreased H3-K4 methylation. TNF-α-induced PASMC proliferation may involve the H3-K4 demethylase enzyme, lysine-specific demethylase 1 (LSD1). Conclusions: TNF-α-induced PASMC proliferation may be partly associated with excessive superoxide formation and histone and DNA methylation.

KDM6A Demethylase Regulates Renal Sodium Excretion and Blood Pressure.

BACKGROUND: KDM6A (Lysine-Specific Demethylase 6A) is a specific demethylase for histone 3 lysine (K) 27 trimethylation (H3K27me3). The purpose of this study is to investigate whether KDM6A in renal tubule cells plays a role in the regulation of kidney function and blood pressure. METHODS: We first crossed Ksp-Cre+/- and KDM6Aflox/flox mice for generating inducible kidney-specific deletion of KDM6A gene. RESULTS: Notably, conditional knockout of KDM6A gene in renal tubule cells (KDM6A-cKO) increased H3K27me3 levels which leads to a decrease in Na excretion and elevation of blood pressure. Further analysis showed that the expression of NKCC2 (Na-K-2Cl cotransporter 2) and NCC (Na-Cl cotransporters) was upregulated which contributes to impaired Na excretion in KDM6A-cKO mice. The expression of AQP2 (aquaporin 2) was also increased in KDM6A-cKO mice, which may facilitate water reabsorption in KDM6A-cKO mice. The expression of Klotho was downregulated while expression of aging markers including p53, p21, and p16 was upregulated in kidneys of KDM6A-cKO mice, indicating that deletion of KDM6A in the renal tubule cells promotes kidney aging. Interestingly, KDM6A-cKO mice developed salt-sensitive hypertension which can be rescued by treatment with Klotho. KDM6A deficiency induced salt-sensitive hypertension likely through downregulation of the Klotho/ERK (extracellular signal-regulated kinase) signaling and upregulation of the WNK (with-no-lysine kinase) signaling. CONCLUSIONS: This study provides the first evidence that KDM6A plays an essential role in maintaining normal tubular function and blood pressure. Renal tubule cell specific KDM6A deficiency causes hypertension due to increased H3K27me3 levels and the resultant downregulation of Klotho gene expression which disrupts the Klotho/ERK/NCC/NKCC2 signaling.

PEGylated porcine-human recombinant uricase: A novel fusion protein with improved efficacy and safety for the treatment of hyperuricemia and renal complications.

The growing prevalence of hyperuricemia necessitates the urgent development of more potent treatments. This study aimed to develop, optimize, and evaluate the safety and efficacy of porcine-human recombinant uricase (PHRU) both in vitro and in vivo. The study employed gene editing of PHRU through site-directed mutagenesis, with recombinant proteins expressed in vitro utilizing Escherichia coli. The polyethylene glycol (PEG) approach was employed to augment uricase stability and diminish immunogenicity. The pharmacokinetics and pharmacodynamics of PHRU were tested in vitro and in Sprague Dawley rats. Successful expression of the fusion protein in E. coli and the development of the PEGylated drug were achieved. In vitro experiments confirmed the efficacy of PEG-PHRU in degrading uric acid, with PEGylation not markedly affecting the biological activity of PHRU. Animal studies revealed that PEG-PHRU significantly lowered plasma uric acid levels and mitigated hyperuricemia-induced renal damage in rats. Both drug metabolism and pharmacokinetics exhibited favorable characteristics without observable adverse effects in experimental animals. This novel fusion protein shows the potential for ameliorating hyperuricemia and related renal complications, highlighting it as a promising drug candidate with substantial market applications.

Distinct response patterns of plants and soil microorganisms to agronomic practices and seasonal variation in a floodplain ecosystem.

Introduction: Climate change and anthropogenic activities are the greatest threats to floodplain ecosystems. A growing body of literature shows that floodplain ecosystems have experienced increased chemical fertilizer and pesticide loads, which will disturb the above and belowground ecosystems. However, we lack knowledge regarding the effects of such human activities on the vegetation and soil microbiomes in these ecosystems. Methods: In the present study, plant functional traits and Illumina Mi-Seq sequencing were to assess the impact of nitrogen fertilizer and glyphosate addition on the structure and function of the vegetation and soil microbiomes (bacteria, fungi, and protists) in a floodplain ecosystem, and to assess the influence of seasonal variation. Results: We identified distinct response mechanisms of plant and microbial communities to the addition of nitrogen fertilizer and glyphosate, and seasonal variation. Nitrogen fertilizer and glyphosate significantly affected plant diversity, aboveground and underground biomass, and C and N content and significantly changed the leaf area and plant stature of dominant plants. However, the addition of nitrogen fertilizer and glyphosate did not significantly affect the diversity and structure of bacterial, fungal, and protist communities. The application of nitrogen fertilizer could improve the negative effects of glyphosate on the functional traits of plant communities. The seasonal variation of floodplain has significantly changed the soil's physical, chemical, and biological properties. Our results showed that compared with that in summer, the soil ecosystem multifunctionality of the floodplain ecosystem in autumn was significantly lower. Seasonal variation had a significant effect on plant diversity and functional traits. Moreover, seasonal variation significantly affected the community compositions, diversity, and structure of bacteria, fungi, and protists. Seasonal variation had a stronger impact on fungal community assembly than on that of bacteria and protists. In summer, the assembly of the fungal community was dominated by a deterministic process, while in autumn, it is dominated by a stochastic process. In addition, the negative association among bacteria, fungi, and protists has been strengthened in autumn and formed a more robust network to cope with external changes. Discussion: These results extended our understanding of the ecological patterns of soil microbiomes in floodplain ecosystems and provided support for enhancing the ecological barrier function and the service potential of floodplain ecosystems.

Adult Mouse Kidney Stem Cells Orchestrate the De Novo Assembly of a Nephron via Sirt2-Modulated Canonical Wnt/ß-Catenin Signaling.

Generation of kidney organoids using autologous kidney stem cells represents an attractive strategy for treating and potentially replacing the failing kidneys. However, whether adult mammalian kidney stem cells have regenerative capacity remains unknown. Here, previously unidentified adult kidney Sca1+ Oct4+ stem/progenitor cells are isolated. Interestingly, culturing these cells leads to generation of kidney-like structures. First, the assembly of self-organizing 3D kidney-like structures is observed. These kidney organoids contain podocytes, proximal tubules, and endothelial cells that form networks of capillary loop-like structures. Second, the differentiation of kidney stem cells into functionally mature tubules and self-organizing kidney-shaped structures in monolayer culture that selectively endocytoses dextran, is shown. Finally, the de novo generation of an entire self-organizing nephron from monolayer cultures is observed. Mechanistically, it is demonstrated that Sirt2-mediated canonical Wnt/ß-catenin signaling is critical for the development of kidney organoids. Thus, the first evidence is provided that the adult mouse kidney stem cells are capable of de novo generating kidney organoids.

Stem cell therapy for pulmonary arterial hypertension: An update.

Pulmonary arterial hypertension (PAH) remains a deadly disease, and there currently is no cure for this life-threating medical problem. The average lifespan is about 5 to 7 years after diagnosis of PAH. Therefore, a conceptual breakthrough to develop new therapeutic strategies for PAH is urgently needed. Growing evidence shows that stem cells are emerging as a novel effective treatment, but the understanding of its underlying mechanisms is still limited. This review highlights the mechanisms through which stem cells successfully reverse pulmonary vascular endothelial dysfunction, pulmonary artery smooth muscle cell over-proliferation, and mitochondrial dysfunction in PAH patients and common rodent models used in PAH research. They can modulate common underlying pathways involved in PAH, including the nitric oxide synthase, mitochondrial regulators, microRNAs and STAT3-BMPR signaling. Genetic modifications further enhance the therapeutic effects of stem cells on PAH. Clinical trials showed promising therapeutic potential of mesenchymal stem cells and endothelial progenitor cells for PAH. Potential limitations and challenges are also discussed. The current findings support the need for further investigation and validation of stem cell therapy for PAH.

Transplantation of bone marrow cells from miR150 knockout mice improves senescence-associated humoral immune dysfunction and arterial stiffness.

BACKGROUND AND PURPOSE: The senescence-accelerated mouse P1 (SAMP1) suffers from humoral immune deficiency, arterial stiffness and accelerated aging. In contrast, the microRNA-150 knockout (miR-150-KO) mice show enhanced humoral immune function including increased B cell population and elevated serum immunoglobulin levels and enjoy extended lifespan. The purpose of this study was to investigate whether transplantation of bone marrow cells (BMCs) from miR-150-KO mice affects immune deficiency and arterial stiffening in SAMP1 mice. METHODS AND RESULTS: Pulse wave velocity and blood pressure were increased significantly in SAMP1 mice (10 months), indicating arterial stiffening and hypertension. Interestingly, transplantation of BMCs from miR-150-KO mice significantly attenuated arterial stiffening and hypertension in SAMP1 mice within eight weeks. BMC transplantation from miR-150-KO mice partially rescued the downregulation of B lymphocytes, largely restored serum IgG and IgM levels, decreased inflammatory cytokine and chemokine expression, and attenuated macrophage and T cell infiltration in aortas in SAMP1 mice. BMC transplantation nearly abolished the upregulation of collagen 1, TGFß1, Scleraxis, MMP-2 and MMP-9 expression and the downregulation of elastin levels in aortas in SAMP1 mice. FISH staining confirmed existence of the transplanted BMCs at end of the experiment. In cultured endothelial cells, IgG-deficient medium invoked upregulation of inflammatory cytokine/chemokine expression which can be rescued by treatment with IgG. CONCLUSIONS: Accelerated senescence caused arterial stiffening via impairing the humoral immune function in SAMP1 mice. BMC transplantation from miR-150-KO mice attenuated arterial matrix remodeling and stiffening and hypertension in SAMP1 mice partly via improving the humoral immune function which attenuates vascular inflammation.

In Vivo Cardiac-specific Expression of Adenylyl Cyclase 4 Gene Protects against Klotho Deficiency-induced Heart Failure.

Klotho is an aging-suppressor gene. Klotho gene deficiency causes heart failure in Klotho-hypomorphic mutant (KL (-/-)) mice. RNA-seq and western blot analysis showed that adenylyl cyclase type IV (AC4) mRNA and protein expression was largely decreased in cardiomyocytes of KL (-/-) mice. The objective of this study was to investigate whether in vivo cardiac-specific expression of AC4 gene protects against Klotho deficiency-induced heart failure. Interestingly, in vivo AAV-based cardiac-specific AC4 gene expression increased left ventricular fractional shortening, ejection fraction, stroke volume, and left ventricular end-diastolic volume in KL (-/-) mice, suggesting that cardiac-specific AC4 gene expression improves Klotho deficiency-induced heart dysfunction. Cardiac-specific AC4 gene expression also decreased Klotho deficiency-induced cardiac hypertrophy. Cardiac-specific AC4 gene expression alleviated Klotho deficiency-induced cardiac fibrosis and calcification. Furthermore, cardiac-specific AC4 gene expression attenuated mitochondrial dysfunction, superoxide accumulation and cardiomyocyte apoptotic cell death. Thus, downregulation of AC4 may contribute to Klotho deficiency-induced heart failure. Mechanistically, AAV2/9-αMHC-AC4 increased cardiomyocytic cAMP levels and thus regulated the PKA-PLN-SERCA2 signal pathway, which is critical in modulating calcium flux and mitochondrial function. In conclusion, cardiac-specific AC4 gene expression protects against Klotho deficiency-induced heart failure through increasing cardiomyocytic cAMP levels, which alleviates cAMP-dependent mitochondrial dysfunction, superoxide accumulation and apoptotic cell death. AC4 regulates superoxide levels via the cAMP-PKA pathway. AC4 could be a potential therapeutic target for heart failure associated with Klotho deficiency. Heart failure is the major cause of mortality in patients with chronic kidney disease (CKD). A decrease in Klotho levels is linked to CKD.

Clinical Progress and Preclinical Insights Into Umbilical Cord Blood Transplantation Improvement.

The application of umbilical cord blood (UCB) as an important source of hematopoietic stem and progenitor cells (HSPCs) for hematopoietic reconstitution in the clinical context has steadily grown worldwide in the past 30 years. UCB has advantages that include rapid availability of donors, less strict HLA-matching demands, and low rates of graft-versus-host disease (GVHD) versus bone marrow (BM) and mobilized peripheral blood (PB). However, the limited number of HSPCs within a single UCB unit often leads to delayed hematopoietic engraftment, increased risk of transplant-related infection and mortality, and proneness to graft failure, thus hindering wide clinical application. Many strategies have been developed to improve UCB engraftment, most of which are based on 2 approaches: increasing the HSPC number ex vivo before transplantation and enhancing HSPC homing to the recipient BM niche after transplantation. Recently, several methods have shown promising progress in UCB engraftment improvement. Here, we review the current situations of UCB manipulation in preclinical and clinical settings and discuss challenges and future directions.

Immunogenicity of the Xcl1-SARS-CoV-2 Spike Fusion DNA Vaccine for COVID-19.

SARS-CoV-2 spike (S) variants that may evade antibody-mediated immunity are emerging. Evidence shows that vaccines with a stronger immune response are still effective against mutant strains. Here, we report a targeted type 1 conventional dendritic (cDC1) cell strategy for improved COVID-19 vaccine design. cDC1 cells specifically express X-C motif chemokine receptor 1 (Xcr1), the only receptor for chemokine Xcl1. We fused the S gene sequence with the Xcl1 gene to deliver the expressed S protein to cDC1 cells. Immunization with a plasmid encoding the S protein fused to Xcl1 showed stronger induction of antibody and antigen-specific T cell immune responses than immunization with the S plasmid alone in mice. The fusion gene-induced antibody also displayed more powerful SARS-CoV-2 wild-type virus and pseudovirus neutralizing activity. Xcl1 also increased long-lived antibody-secreting plasma cells in bone marrow. These preliminary results indicate that Xcl1 serves as a molecular adjuvant for the SARS-CoV-2 vaccine and that our Xcl1-S fusion DNA vaccine is a potential COVID-19 vaccine candidate for use in further translational studies.