Heparanase promotes the onset and progression of atherosclerosis in apolipoprotein E gene knockout mice.

BACKGROUND AND AIMS: Atherosclerosis is the primary underlying cause of myocardial infarction and stroke, which are the major causes of death globally. Heparanase (Hpse) is a pro-inflammatory extracellular matrix degrading enzyme that has been implicated in atherogenesis. However, to date the precise roles of Hpse in atherosclerosis and its mechanisms of action are not well defined. This study aims to provide new insights into the contribution of Hpse in different stages of atherosclerosis in vivo. METHODS: We generated Hpse gene-deficient mice on the atherosclerosis-prone apolipoprotein E gene knockout (ApoE-/-) background to investigate the impact of Hpse gene deficiency on the initiation and progression of atherosclerosis after 6 and 14 weeks high-fat diet feeding, respectively. Atherosclerotic lesion development, blood serum profiles, lesion composition and aortic immune cell populations were evaluated. RESULTS: Hpse-deficient mice exhibited significantly reduced atherosclerotic lesion burden in the aortic sinus and aorta at both time-points, independent of changes in plasma cholesterol levels. A significant reduction in the necrotic core size and an increase in smooth muscle cell content were also observed in advanced atherosclerotic plaques of Hpse-deficient mice. Additionally, Hpse deficiency reduced circulating and aortic levels of VCAM-1 at the initiation and progression stages of disease and circulating MCP-1 levels in the initiation but not progression stage. Moreover, the aortic levels of total leukocytes and dendritic cells in Hpse-deficient ApoE-/- mice were significantly decreased compared to control ApoE-/-mice at both disease stages. CONCLUSIONS: This study identifies Hpse as a key pro-inflammatory enzyme driving the initiation and progression of atherosclerosis and highlighting the potential of Hpse inhibitors as novel anti-inflammatory treatments for cardiovascular 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.

Heparanase Deficiency Is Associated with Disruption, Detachment, and Folding of the Retinal Pigment Epithelium.

PURPOSE: Pentosan polysulfate sodium (PPS; Elmiron) is a FDA-approved heparanase inhibitor for the treatment of bladder pain and interstitial cystitis. The chronic use of PPS has been associated with a novel pigmentary maculopathy, associated with discrete vitelliform deposits that exhibit hyperfluorescence, macular hyper-pigmentary spots, and foci of nodular RPE enlargement. Therefore, this study aimed to investigate the retinal morphology of heparanase knockout mice. MATERIAL AND METHODS: The retinal morphology of heparanase knock-out and age-matched control wild type mice of 3-, 9- and 15-weeks old was characterized by means of histological evaluation. Immuno-histological stains for RPE65, F4/80 and Ki67 were performed for investigating the RPE, inflammatory and proliferating cells, respectively. RESULTS: Histological analysis showed no changes in age-matched wild-type controls, whereas the eyes of heparanase null mice were characterized by alterations in RPE and neural retina, as manifest by RPE folds and choroidal thickening, detached RPE cells, thickening of the photoreceptor layer and retinal disorganization. The presence of discrete hyperfluorescent foci, however, was absent. The prevalence of the RPE/choroidal changes or protrusions seemed to progress over time and were correlated with more RPE65 signal rather than influx of F4/80- or Ki67-positive cells. These results indicate that the subretinal alterations were mostly RPE driven, without influx of inflammatory or proliferating cells. CONCLUSIONS: Our results indicate that heparanase deficiency in the mice leads to RPE folds, choroidal thickening, and retinal disorganization. The presence of discrete hyperfluorescent foci, a key characteristic of the human disease, was not observed. However, it can be concluded that some of the observations in mice are similar to those seen after chronic use of PPS in humans. These findings indicate that the toxicity observed in the presence of heparanase inhibitors is target-related and will preclude the clinical use of heparanase inhibition as a therapeutic intervention.

Analysis of Immune Responses in Acinetobacter baumannii-Infected Klotho Knockout Mice: A Mouse Model of Acinetobacter baumannii Infection in Aged Hosts.

Acinetobacter baumannii is an important opportunistic pathogen that primarily afflicts elderly people. To clarify the pathogenicity of A. baumannii in the elderly, we investigated immune responses to A. baumannii ATCC 19606 infection in klotho knockout (KO) mice, the mouse model of aging. Following intravenous inoculation, the mice seldom displayed severe symptoms. However, the survival rate was 56% at 7 days post-infection. Bacteria were detected in the lungs of klotho KO mice but not klotho wildtype (WT) mice at 7 days post-infection. Neutrophils, eosinophils, interstitial macrophages, and monocyte/dendritic cell subset in the lungs of klotho KO mice were transiently induced after infection with A. baumannii. The number of alveolar macrophages in klotho KO mice was lower than that in klotho WT mice, except for 1 day post-infection. CD11b expression on neutrophils and alveolar macrophages in the lungs of klotho KO mice was seldom upregulated by the infection. These results suggested that immune functions eliminating bacteria in the lungs of klotho KO mice were insufficient. CD11blow conventional DC cells hardly increased in klotho KO mice infected with A. baumannii. Additionally, the production of interleukin (IL)-10 in the sera of klotho KO mice was significantly higher than that in klotho WT mice, whereas that production of interferon-gamma was not detected in the sera of klotho KO mice. These results suggested that acquired immune responses were hardly induced in klotho KO mice. IL-1ß, CXCL1, CXCL2, and CCL2 expression was significantly higher in the lungs of klotho KO mice infected with A. baumannii than in those of klotho WT mice at 1 day post-infection. These results suggested that pulmonary inflammation was elicited in klotho KO mice during early infection. The expression levels of proinflammatory cytokines significantly correlated with TLR9 expression in the lungs of klotho KO mice. The collective results demonstrate an A. baumannii infection state in aged hosts and suggest that pulmonary inflammation and bacterial burden should be noted in aged hosts even in the absence of severe symptoms of A. baumannii infection.

Klotho deficiency aggravates diabetes-induced podocyte injury due to DNA damage caused by mitochondrial dysfunction.

Diabetic nephropathy (DN) is a progressive disease, the main pathogeny of which is podocyte injury inducing glomerular filtration barrier and proteinuria. The occurrence and development of DN could be partly attributed to the reactive oxygen species (ROS) generated by mitochondria. However, research on how mitochondrial dysfunction (MtD) ultimately causes DNA damage is poor. Here, we investigated the influence of Klotho deficiency on high glucose (HG)-induced DNA damage in vivo and in vitro. First, we found that the absence of Klotho aggravated diabetic phenotypes indicated by podocyte injury accompanied by elevated urea albumin creatinine ratio (UACR), creatinine and urea nitrogen. Then, we further confirmed that Klotho deficiency could significantly aggravate DNA damage by increasing 8-OHdG and reducing OGG1. Finally, we demonstrated Klotho deficiency may promote MtD to promote 8-OHdG-induced podocyte injury. Therefore, we came to a conclusion that Klotho deficiency may promote diabetes-induced podocytic MtD and aggravate 8-OHdG-induced DNA damage by affecting OOG1.

Genetic background influences the impact of KLOTHO deficiency.

Genetic background is a key but sometimes overlooked factor that profoundly impacts disease susceptibility and presentation in both humans and disease models. Here we show that deficiency of KLOTHO protein, an important renal regulator of mineral homeostasis and a cofactor for FGF23, causes different phenotypes in 129S1/SvlmJ (129) and C57BL/6J (B6) mouse strains. The 129 strain is more severely affected, with decreased longevity, decreased body weight, and increased amounts of kidney calcification compared with B6 mice. Reciprocal F1 crosses of the strains also indicate a parentage effect on the Klotho phenotype with F1 KLOTHO-deficient progeny of B6 mothers and 129 fathers having more kidney calcification than progeny of 129 mothers and B6 fathers. Comparing and contrasting the genetic architecture leading to different phenotypes associated with specific inbred mouse strains may reveal previously unrecognized and important metabolic interactions affecting chronic kidney disease.

Endocrine fibroblast growth factors as potential biomarkers for chronic kidney disease.

INTRODUCTION: Among the family of fibroblast growth factors (FGFs), FGF19, FGF21, and FGF23 act as circulating hormones and are called endocrine FGFs. FGF19 and FGF21 regulate bile acid and energy homeostasis, respectively, whereas FGF23 regulates vitamin D and phosphate homeostasis. Accumulating evidence suggests that FGF23 plays a critical role in disturbed mineral metabolisms, left ventricular hypertrophy, immunosuppression, inflammation, among others in patients with chronic kidney disease (CKD), highlighting the potential both as a biomarker and a therapeutic target. Several studies have also examined the potential role of FGF19 and FGF21 in CKD patients. AREAS COVERED: In this review, we present a brief overview of the biology of FGF19, FGF21, and FGF23, and summarize recent clinical and experimental studies on the pathophysiological roles of endocrine FGFs, mainly FGF23, in CKD patients. EXPERT OPINION: Among the endocrine FGFs, FGF23 represents the most promising biomarker in CKD patients. If future studies confirm that FGF23 is directly toxic in CKD patients, FGF23 could be regarded as a therapeutic target and its measurement would be valuable if applied in clinical practice. Despite their potentially important roles, the clinical relevance of FGF19 and FGF21 in CKD patients is unclear, and much more studies are required.

Salt causes aging-associated hypertension via vascular Wnt5a under Klotho deficiency.

Aging is associated with a high prevalence of hypertension due to elevated susceptibility of BP to dietary salt, but its mechanism is unknown. Serum levels of Klotho, an anti-aging factor, decline with age. We found that high salt (HS) increased BP in aged mice and young heterozygous Klotho-knockout mice and was associated with increased vascular expression of Wnt5a and p-MYPT1, which indicate RhoA activity. Not only the Wnt inhibitor LGK974 and the Wnt5a antagonist Box5 but Klotho supplementation inhibits HS-induced BP elevation, similarly to the Rho kinase inhibitor fasudil, associated with reduced p-MYPT1 expression in both groups of mice. In cultured vascular smooth muscle cells, Wnt5a and angiotensin II (Ang II) increased p-MYPT1 expression but knockdown of Wnt5a with siRNA abolished Ang II-induced upregulation of p-MYPT1, indicating that Wnt5a is indispensable for Ang II-induced Rho/ROCK activation. Notably, Klotho inhibited Wnt5a- and Ang II-induced upregulation of p-MYPT1. Consistently, Klotho supplementation ameliorated HS-induced augmentation of reduced renal blood flow (RBF) response to intra-arterial infusion of Ang II and the thromboxane A2 analog U46619, which activated RhoA in both groups of mice and were associated with the inhibition of BP elevation, suggesting that abnormal response of RBF to Ang II contributes to HS-induced BP elevation. Thus, Klotho deficiency underlies aging-associated salt-sensitive hypertension through vascular non-canonical Wnt5a/RhoA activation.

Serum Klotho, Cardiovascular Events, and Mortality in Nondiabetic Chronic Kidney Disease.

BACKGROUND: Experimental studies indicate that Klotho deficiency is a pathogenic factor for CKD-related complications, including cardiovascular disease (CVD). However, the association between serum Klotho and clinical outcomes in nondiabetic CKD patients needs to be further clarified. We aimed to determine whether serum Klotho levels are associated with CVD events and mortality in predialysis CKD patients without diabetes. METHODS: A total of 336 CKD stage 2-5 predialysis patients without diabetes were recruited and followed from the end of 2014 to January 2019 for CVD events and overall mortality. Serum Klotho was detected by ELISA and divided into quartiles (lowest, middle, second highest, and highest quartiles) according to their serum Klotho category. RESULTS: After a median follow-up of 3.52 years (IQR 3.34-3.76), Kaplan-Meier analysis showed that, compared to participants with a Klotho level in the highest quartile (the reference category), those in the lowest Klotho quartile were associated with a higher all-cause mortality risk (HR = 7.05; 95% CI 1.59-31.25) and a higher CVD event risk (HR = 3.02; 95% CI 1.45-6.30). In addition, the middle Klotho quartile was also associated with CVD event risk (HR = 2.56; 95% CI 1.21-5.41). Moreover, in the multivariate-adjusted model, the lowest Klotho quartile remained significantly associated with all-cause mortality (HR = 5.17; 95% CI 1.07-24.96), and the middle Klotho quartile maintained a significant association with CVD event risk (HR = 2.32; 95% CI 1.03-5.21). CONCLUSION: These results suggest that lower serum Klotho levels are independently associated with overall mortality and CVD events in nondiabetic predialysis CKD patients.

Epigenetic Regulation of KL (Klotho) via H3K27me3 (Histone 3 Lysine [K] 27 Trimethylation) in Renal Tubule Cells.

KL (klotho) levels decline with age, which is an important mechanistic driver of aging. KL gene deficiency is associated with hypertension. The purpose of this study is to investigate the potential role of H3K27me3 (histone 3 lysine [K] 27 trimethylation) in the regulation of KL gene expression and examine the related molecular pathways that may drive kidney cell aging. Kidneys were collected from 6-month-old WT (wild type; young WT), 30-month-old WT (aged WT), and 6- (young) and 20-month-old (aged) KL mutant mice, respectively. We demonstrated that the H3K27me3 level was increased in kidneys of aged WT and KL mutant mice versus young WT mice. Elevation of H3K27me3 levels was likely due to downregulation of the H3K27 (histone H3 Lys 27)-specific demethylase JMJD3 (the Jumonji domain containing-3) in the aged kidneys. Inhibition of PRC2 (polycomb repressive complex C2; histone trimethyltransferase) decreased the H3K27me3 levels leading to an increase in the expression of KL in cultured primary renal tubule cells assessed by Western blot and KL promoter activity assays. The chromatin immunoprecipitation qPCR assay revealed that H3K27me3 was physically associated with the KL promoter region. Furthermore, aging impaired the SGK1 (serum- and glucocorticoid-induced protein kinase 1)/FOXO3a (the forkhead box class O 3a) signaling leading to upregulation of p53 and p16 (aging markers) in the kidney of aged WT mice. KL may regulate the SGK1/FOXO3 signaling, which was decreased due to KL deficiency. Thus, aging-associated downregulation of KL gene expression may be partly attributed to upregulation of H3K27me3 levels. Downregulation of KL may impair the SGK1/FOXO3 signaling contributing to kidney cell aging.

Decreased ADAM17 expression in the lungs of α-Klotho reduced mouse.

The deficiency of α-Klotho in mice causes phenotypes resembling human age-associated disorders at 3-4 weeks after birth and shows short lifespans of ∼2 months. One of the crucial symptoms is pulmonary emphysema, although α-Klotho is not expressed in the lungs. α-Klotho secreted from the kidneys is probably involved in the pathology of emphysema because kidney-specific knockout mice exhibit emphysematous structural changes. We examined whether any glycan changes in α-Klotho mouse lungs were observed, because α-Klotho is reported to have glycosidase activity. Here, we found the accumulation of heparan sulphate in the microsomal fraction of α-Klotho mouse lungs. Meanwhile, a disintegrin and metalloproteinase 17 (ADAM17) expression was decreased in α-Klotho mice. From these results, it is thought that the increase in heparan sulphate is due to insufficient cleavage of the core protein by ADAM17. Additionally, a reduction in α-Klotho and a decline of ADAM17 were also observed both in normal aged mice and in senescence marker protein-30 (SMP30) knockout mice, a mouse model of premature ageing. Thus, the decrease in ADAM17 is caused by the reduction in α-Klotho. These may be involved in the deterioration of lung function during ageing and may be associated with the pathology of pulmonary emphysema.

Magnesium prevents vascular calcification in Klotho deficiency.

Klotho knock-out mice are an important model for vascular calcification, which is associated with chronic kidney disease. In chronic kidney disease, serum magnesium inversely correlates with vascular calcification. Here we determine the effects of serum magnesium on aortic calcification in Klotho knock-out mice treated with a minimal or a high magnesium diet from birth. After eight weeks, serum biochemistry and aorta and bone tissues were studied. Protective effects of magnesium were characterized by RNA-sequencing of the aorta and micro-CT analysis was performed to study bone integrity. A high magnesium diet prevented vascular calcification and aortic gene expression of Runx2 and matrix Gla protein found in such mice on the minimal magnesium diet. Differential expression of inflammation and extracellular matrix remodeling genes accompanied the beneficial effects of magnesium on calcification. High dietary magnesium did not affect serum parathyroid hormone, 1,25-dihydroxyvitamin D3 or calcium. High magnesium intake prevented vascular calcification despite increased fibroblast growth factor-23 and phosphate concentration in the knock-out mice. Compared to mice on the minimal magnesium diet, the high magnesium diet reduced femoral bone mineral density by 20% and caused excessive osteoid formation indicating osteomalacia. Osteoclast activity was unaffected by the high magnesium diet. In Saos-2 osteoblasts, magnesium supplementation reduced mineralization independent of osteoblast function. Thus, high dietary magnesium prevents calcification in Klotho knock-out mice. These effects are potentially mediated by reduction of inflammatory and extracellular matrix remodeling pathways within the aorta. Hence magnesium treatment may be promising to prevent vascular calcification, but the risk for osteomalacia should be considered.

Autophagy plays a critical role in Klotho gene deficiency-induced arterial stiffening and hypertension.

Klotho is an anti-aging gene that shortens the life span when disrupted and extends the lifespan when overexpressed. This study investigated whether autophagy plays a role in Klotho gene deficiency-induced arterial stiffening and hypertension. Klotho mutant heterozygous (KL+/-) mice and age- and sex-matched wild-type (WT) mice were used. Arteries were examined for autophagy using Western blot assays. Pulse wave velocity (PWV), a direct measure of arterial stiffness, and blood pressure (BP) increased significantly in KL (+/-) mice. The autophagy level, as measured by LC3-II expression and autophagy flux, increased in aortas of KL (+/-) mice, indicating that Klotho gene deficiency upregulated autophagy. Chloroquine diminished Klotho gene deficiency-induced increases in PWV and BP and eliminated the upregulation of autophagic flux in KL (+/-) mice. Klotho gene deficiency-induced arterial stiffness was accompanied by upregulation of MMP9, TGFß-1, TGFß-3, RUNX2, and ALP, but these changes were effectively mitigated by chloroquine. Chloroquine also halted an increase in scleraxis expression in aortas of Klotho (+/-) mice. In cultured mouse aortic smooth muscle cells, Klotho gene deficiency increased autophagy, leading to upregulation of scleraxis, a key transcription factor of collagen synthesis. Klotho gene deficiency failed to upregulate scleraxis expression when autophagy was inhibited, suggesting that autophagy is a critical mediator of Klotho gene deficiency-induced upregulation of scleraxis. Suppression of enhanced autophagy by chloroquine lessens Klotho gene deficiency-induced arterial stiffening and hypertension by stopping upregulation of MMP9 and scleraxis. The enhanced autophagic activity plays a crucial role in Klotho gene deficiency-induced arterial stiffening and hypertension. KEY MESSAGES: Klotho gene deficiency upregulates autophagy. Upregulation of autophagy plays a role in the pathogenesis of arterial stiffening. Autophagy regulates MMP9 activity and scleraxis expression.

Heparanase Accelerates Obesity-Associated Breast Cancer Progression.

Obese women have higher risk of bearing breast tumors that are highly aggressive and resistant to therapies. Tumor-promoting effects of obesity occur locally via adipose inflammation and related alterations to the extracellular matrix (ECM) as well as systemically via circulating metabolic mediators (e.g., free fatty acids, FFA) associated with excess adiposity and implicated in toll-like receptor-mediated activation of macrophages-key cellular players in obesity-related cancer progression. Although the contribution of macrophages to proneoplastic effects of obesity is well documented, the role of ECM components and their enzymatic degradation is less appreciated. We show that heparanase, the sole mammalian endoglucuronidase that cleaves heparan sulfate in ECM, is preferentially expressed in clinical/experimental obesity-associated breast tumors. Heparanase deficiency abolished obesity-accelerated tumor progression in vivo. Heparanase orchestrated a complex molecular program that occurred concurrently in adipose and tumor tissue and sustained the cancer-promoting action of obesity. Heparanase was required for adipose tissue macrophages to produce inflammatory mediators responsible for local induction of aromatase, a rate-limiting enzyme in estrogen biosynthesis. Estrogen upregulated heparanase in hormone-responsive breast tumors. In subsequent stages, elevated levels of heparanase induced acquisition of procancerous phenotype by tumor-associated macrophages, resulting in activation of tumor-promoting signaling and acceleration of breast tumor growth under obese conditions. As techniques to screen for heparanase expression in tumors become available, these findings provide rational and a mechanistic basis for designing antiheparanase approaches to uncouple obesity and breast cancer in a rapidly growing population of obese patients. SIGNIFICANCE: This study reveals the role of heparanase in promoting obesity-associated breast cancer and provides a mechanistically informed approach to uncouple obesity and breast cancer in a rapidly growing population of obese patients.

Klotho inhibits PKCα/p66SHC-mediated podocyte injury in diabetic nephropathy.

Diabetic nephropathy (DN) is a progressive disease, the main pathogeny of which is podocyte injury. As a calcium-dependent serine/threonine protein kinase involved in podocyte injury, protein kinase C isoform α (PKCα) was reported to regulate the phosphorylation of p66SHC. However, the role of PKCα/p66SHC in DN remains unknown. Klotho, an anti-aging protein with critical roles in protecting kidney, is expressed predominantly in the kidney and secreted in the blood. Nonetheless, the mechanism underlying amelioration of podocyte injury by Klotho in DN remains unclear. Our data showed that Klotho was decreased in STZ-treated mice and was further declined in diabetic KL ± mice. As expected, Klotho deficiency aggravated diabetes-induced proteinuria and podocyte injury, accompanied by the activation of PKCα and p66SHC. In contrast, overexpression of Klotho partially ameliorated PKCα/p66SHC-mediated podocyte injury and proteinuria. In addition, in vitro experiments showed that activation of PKCα and subsequently increased intracellular reactive oxygen species (ROS) was involved in podocytic apoptosis induced by high glucose (HG), which could be partially reversed by Klotho. Hence, we conclude that Klotho might inhibit PKCα/p66SHC-mediated podocyte injury in diabetic nephropathy.

Multifaceted Mechanisms of Vascular Calcification in Aging.

Approximately 20% of the world's population will be around or above 65 years of age by the next decade. Out of these, 40% are suspected to have cardiovascular diseases as a cause of mortality. Arteriosclerosis, characterized by increased vascular calcification, impairing Windkessel effect and tissue perfusion, and determining end-organ damage, is a hallmark of vascular pathology in the elderly population. Risk factors accumulated during aging affect the normal physiological and vascular aging process, which contributes to the progression of arteriosclerosis. Traditional risk factors, age-associated diseases, and respective regulating mechanisms influencing vascular calcification and vascular stiffness have been extensively studied for many years. Despite the well-known fact that aging alone can induce vascular damage, specific mechanisms that implicate physiological aging in vascular calcification, contributing to vascular stiffness, are poorly understood. This review focuses on mechanisms activated during normal aging, for example, cellular senescence, autophagy, extracellular vesicles secretion, and oxidative stress, along with the convergence of premature aging models' pathophysiology, such as Hutchinson-Gilford Progeria (prelamin accumulation) and Klotho deficiency, to understand vascular calcification in aging. Understanding the mechanisms of vascular damage in aging that intersect with age-associated diseases and risk factors is crucial to foster innovative therapeutic targets to mitigate cardiovascular disease. Visual Overview- An online visual overview is available for this article.

Klotho deficiency affects the spine morphology and network synchronization of neurons.

Klotho-deficient mice rapidly develop cognitive impairment and show some evidence of the onset of neurodegeneration. However, it is impossible to investigate the long-term consequences on the brain because of the dramatic shortening of lifespan caused by systemic klotho deficiency. As klotho expression is downregulated with advancing organismal age, understanding the mechanisms of klotho action is important for developing novel strategies to support healthy brain aging. Previously, we reported that klotho-deficient mice show enhanced long-term potentiation prior to the onset of cognitive impairment. To inform this unusual phenotype, herein, we examined neuronal structure and in vitro synaptic function. Our results indicate that klotho deficiency causes the population of dendritic spines to shift towards increased head diameter and decreased length consistent with mature, mushroom type spines. Multi-electrode array recordings from klotho-deficient neurons show increased synchronous firing and activity changes reflective of increased neuronal network activity. Supplementation of the neuronal growth media with recombinant shed klotho corrected some but not all of the activity changes caused by klotho deficiency. Last, in vivo we found that klotho-deficient mice have a decreased latency to induced seizure activity. Together these data show that klotho-deficient memory impairments are underpinned by structural and functional changes that may preclude ongoing normal cognition.

Vestronidase Alfa: A Review in Mucopolysaccharidosis VII.

Mucopolysaccharidosis VII is an extremely rare, autosomal recessive lysosomal storage disorder characterized by a deficiency of ß-glucuronidase activity, resulting in partial degradation and accumulation of GAGs in numerous tissues throughout the body, with consequent cellular damage and organ dysfunction. Enzyme replacement therapy (ERT) with intravenous vestronidase alfa (Mepsevii™), a recombinant form of human ß-glucuronidase, is the first disease-specific therapy approved for the treatment of mucopolysaccharidosis VII in pediatric and adult patients. In the pivotal, blind start, phase 3 trial, 24 weeks of vestronidase alfa therapy significantly reduced urinary GAG (uGAG) excretion in patients with mucopolysaccharidosis VII. Based on a Multi-Domain Responder Index (MDRI; comprises six clinically important morbidity domains, with prespecified minimally important differences for each domain), most evaluable patients experienced an improvement in ≥ 1 domain during the 24-week primary assessment period (overall positive mean change of 0.5 domains). The clinical benefits of vestronidase alfa were sustained during longer-term treatment, as was the reduction in uGAG excretion. Vestronidase alfa has a manageable tolerability profile, with most adverse reactions of mild to moderate severity. Given the lack of treatment options and the clinical benefits it provides, intravenous vestronidase alfa is an important emerging ERT for patients with mucopolysaccharidosis VII.

Klotho Deficiency Accelerates Stem Cells Aging by Impairing Telomerase Activity.

Understanding the effect of molecular pathways involved in the age-dependent deterioration of stem cell function is critical for developing new therapies. The overexpression of Klotho (KL), an antiaging protein, causes treated animal models to enjoy extended life spans. Now, the question stands: Does KL deficiency accelerate stem cell aging and telomere shortening? If so, what are the specific mechanisms by which it does this, and is cycloastragenol (CAG) treatment enough to restore telomerase activity in aged stem cells? We found that KL deficiency diminished telomerase activity by altering the expression of TERF1 and TERT, causing impaired differentiation potential, pluripotency, cellular senescence, and apoptosis in stem cells. Telomerase activity decreased with KL-siRNA knockdown. This suggests that both KL and telomeres regulate the stem cell aging process through telomerase subunits TERF1, POT1, and TERT using the TGFß, Insulin, and Wnt signaling. These pathways can rejuvenate stem cell populations in a CD90-dependent mechanism. Stem cell dysfunctions were largely provoked by KL deficiency and telomere shortening, owing to altered expression of TERF1, TGFß1, CD90, POT1, TERT, and basic fibroblast growth factor (bFGF). The CAG treatment partially rescued telomerase deterioration, suggesting that KL plays a critical role in life-extension by regulating telomere length and telomerase activity.

Theanine attenuates memory impairments induced by klotho gene depletion in mice.

Theanine (γ-glutamylethylamide), an amino acid in tea, is a putative neuroprotective and antioxidant compound capable of improving lifespan and cognitive function. Because we previously reported cognitive dysfunction in klotho mutant mice via down-regulation of janus kinase 2 (JAK2) and signal transducer and activator of transcription3 (STAT3), M1 muscarinic cholinergic receptor (M1 mAChR), and ERK signaling, we, therefore, investigated whether self-administration of theanine affects memory dysfunction in response to klotho gene depletion in mice, and whether theanine modulates the JAK2/STAT3, M1 mAChR, and ERK signaling network. Theanine significantly attenuated memory impairments in klotho mutant mice. Moreover, theanine self-administration significantly attenuated inhibitions of JAK2/STAT3 phosphorylation, M1 mAChR expression, and ERK1/2 phosphorylation in the hippocampus of klotho mutant mice. Consistently, AG490, a JAK2/STAT3 inhibitor, dicyclomine, an M1 mAChR antagonist, or U0126, an ERK1/2 inhibitor, significantly counteracted theanine-induced attenuation of memory impairment induced by klotho gene depletion in mice. Our study suggests that theanine attenuates memory impairments in a genetic aging model via up-regulation of JAK2/STAT3, M1 mAChR, and ERK signaling.