[Sleep disturbance in elderly patients with dementia at home and the factors associated with its identification: An observational study].

AIMS: The purpose of this study was to objectively quantify the sleep of elderly patients with dementia at home using a device and to investigate the factors associated with its identification. METHODS: Sixteen patients (6 males [37.5%], 84.1±4.7 years old; and 10 patients with mild dementia [62.5%]) and their family caregivers who were using outpatient memory clinics and home-visiting nursing station in Japan were included. Demographic and clinical data of the patients and their family caregivers, subjective perceptions of patients' sleep, family caregivers' Zarit care burden, and whether or not they were aware of patients' sleep problems were determined. Nighttime sleep parameters were collected for one week using a non-wearable actigraph. Sleep parameters were compared with patients' subjective views and family caregivers' observations to investigate factors indicative of sleep disturbance. RESULTS: Nighttime sleep parameters for 1 week (mean) were follows: sleep efficiency, 77.2%±9.3%; asleep time, 442.3±99.9 minutes; sleep latency, 18.2±15.8 minutes; awake time, 105.1±69.7 minutes; and number of times leaving the bed, 4.6±3.8 (maximum of 29/night). A significant positive correlation was found between sleep efficiency and duration of dementia (r=0.53, p=0.046), while no correlation was found with dementia severity or Zarit care burden score. The agreement between the patients' complaints about sleep and sleep efficiency (75%) was 30.7%, and family caregivers' awareness of patients' nighttime awakening and bed-leaving was significantly associated with patients' incontinence (p=0.024) and a greater dementia severity (p=0.027). CONCLUSIONS: Elderly dementia patients experienced sleep disturbance at home, such as nighttime awakening and associated bed-leaving; however, it might be difficult to identify these patients at an early stage based on their own complaints and observations by family caregivers. Identifying sleep problems at an early stage may thus require the use of objective measurement devices.

Anti-oxidative effect of Klotho on endothelial cells through cAMP activation.

Klotho, a regulatory factor implicated in countering the aging process, has been reported to ameliorate endothelial dysfunction in vivo. To clarify whether Klotho protein directly affects endothelial cell function, we studied the effects of membrane-form Klotho on manganese superoxide dismutase (Mn-SOD) expression and nitric oxide production in human umbilical vein endothelial cells (HUVEC). We incubated HUVEC with conditioned medium from COS-1 cells transfected with expression vector, pCAGGS-klotho (Klotho-CM) or a recombinant, purified 6His-tagged Klotho protein. Both Klotho-CM and 6His-tagged Klotho protein enhanced Mn-SOD expression by approximately two-fold, partially via activation of the cAMP signaling pathway. Furthermore, Klotho-CM increased nitric oxide production, which also contributed to the up-regulation of Mn-SOD. Using the oxidation-sensitive dye dihydroethidium, we found that Klotho inhibited angiotensin II-induced reactive oxygen species production in HUVEC. These findings provide new insights into the mechanisms of Klotho action and support the therapeutic potential of membrane-form Klotho to regulate endothelial function.

Matrix extracellular phosphoglycoprotein (MEPE) is highly expressed in osteocytes in human bone.

The matrix extracellular phosphoglycoprotein (MEPE) gene is highly expressed in tumors that cause oncogenic hypophosphatemic osteomalacia (OHO). MEPE is also known as one of the bone-tooth matrix proteins and is associated with bone mineralization. We developed a rabbit polyclonal antibody directed against recombinant human MEPE (rhMEPE) after cloning its cDNA from the cDNA library of a nasal tumor tissue causing OHO. Using this antibody, we analyzed the distribution of MEPE in human bones by immunohistochemistry. In bone specimens from normal subjects, MEPE was predominantly expressed by osteocytes and not by osteoblasts. In bone specimens from patients with osteomalacia, however, MEPE was focally expressed by deeply located osteocytes. We also compared the MEPE positivity of osteocytes in mineralized bone and non-mineralized osteoid obtained from patients with osteomalacia and osteoporosis. Among osteomalacia patients, MEPE positivity was seen in 87.5 +/- 8.6% of the osteocytes from mineralized bone compared with 7.8 +/- 6.4% of those from osteoid. Among osteoporosis patients, MEPE positivity was found in 95.3 +/- 0.5% of the osteocytes from mineralized bone compared with 4.9 +/- 5.7% of those from osteoid. MEPE was mainly expressed by osteocytes embedded in the matrix of mineralized bone from patients with osteomalacia or osteoporosis. Our data provide the first histological evidence that MEPE is predominantly expressed by osteocytes in human bone, with significant expression by osteocytes within mineralized bone.

Insulin-mediated regulation of the endothelial renin-angiotensin system and vascular cell growth.

OBJECTIVE: Insulin has a growth-stimulating effect for vascular tissue. At the tissue level, the vascular renin-angiotensin system (RAS) may be involved in the progression of atherosclerosis or vascular hypertrophy. We previously reported that the vascular RAS activity is activated in vascular smooth muscle cells (SMC) by insulin stimulation. However, the effect of insulin on the RAS in endothelial cells (EC) is not fully understood. METHODS: Cultured human EC were incubated with or without insulin. After incubation for 48 h, cellular angiotensinogen and renin mRNA expression and levels in the cells were quantified by slot-blot hybridization and radioimmunoassay. Angiotensin I converting enzyme (ACE) activity in EC homogenates was measured by modified Cushman and Cheung method. EC growth and SMC with or without EC using co-culture were assessed by 3H-thymidine uptake for evaluation of their growth. RESULTS: All doses of insulin (10, 100, 1000 microU/ml) decreased angiotensinogen and renin mRNA expression (angiotensinogen: 19.3%, P < 0.05; 25.4%, P < 0.01; 26.2%, P < 0.01, renin: 12.9%, P < 0.05; 21.3%, P < 0.01; 14.3%, P < 0.05, respectively). Both cellular angiotensinogen and renin level were also reduced by high levels of insulin. Neither 10 nor 100 microU/ml insulin increased cellular angiotensin converting enzyme (ACE) activity (2.17 to 3.48-folds, P = 0.077, 0.125, respectively) significantly, but 1000 microU/ml insulin strongly up-regulated ACE activity by 16.67-folds (P = 0.001) in cultured EC. For the co-culture with EC and SMC, 100 microU/ml insulin was not able to induce SMC but 1000 microU/ml insulin accelerated SMC growth in the co-culture. In contrast insulin that was over 100 microU/ml induced SMC growth in the sole culture of SMC. CONCLUSION: Either low or high levels of insulin suppressed angiotensinogen and renin expression, however, high doses of insulin stimulated ACE activity in cultured human aortic EC. This may indicate that insulin regulates vascular cell growth and endothelial function via bifunctional modification of the vascular angiotensin generation.

Klotho protein activates the PKC pathway in the kidney and testis and suppresses 25-hydroxyvitamin D3 1alpha-hydroxylase gene expression.

Homozygous Klotho mutant (kl-/-) mice exhibit a variety of phenotypes resembling human aging, including arteriosclerosis, infertility, skin atrophy, osteoporosis, and short life span. Calcium abnormality, one of the phenotypes in kl-/- mice, is thought to be due to the elevated gene expression of 25-hydroxyvitamin D3 1alpha-hydroxylase in the kidney. We studied 25-hydroxy-vitamin D3 1alpha-hydroxylase gene expression using a Klotho plasmid that we had previously constructed for Klotho protein production. It was found that Klotho protein medium upregulated cAMP and the PKC pathway, and suppressed 25-hydroxyvitamin D3 1alpha-hydrox-ylase in kidney cells. However, both cAMP and PKC are known to elevate 25-hydroxyvitamin D3 1alpha-hydroxylase gene expression, therefore, another unknown calcium regulation pathway using Klotho protein medium might exist. Furthermore, we found that activation of the PKC pathway by Klotho was observed only in the kidney and testis, where the Klotho gene is expressed, although activation of the cAMP pathway was observed in any kind of cell. These data suggest that calcium regulation through 25-hydroxyvitamin D3 1alpha-hydroxylase by Klotho depends on non-cAMP and a non-PKC pathway and that the Klotho protein may have different signaling pathways, depending on the Klotho gene expression in different cells and organs.

Role of endothelin-1 induced by insulin in the regulation of vascular cell growth.

Insulin is not only a growth factor for vascular cells, but also an inducer of other vasoactive substances such as endothelin-1 (ET-1) in vascular cells. The aim of the present study was to assess the role of endothelial cells (EC) in insulin mediated vascular smooth muscle cell (VSMC) proliferation. Cultured human aortic EC and VSMC were separately incubated. EC were stimulated with insulin (0 to 1000 microU/mL) for 24 h, in the presence or absence of anti-insulin-growth factor-1 (anti-IGF-1) receptor antibody (alphaIR(3)) or a nonselective ET-1 receptor antagonist (TAK044). Cell proliferation was measured by determining (3)H-thymidine uptake. Although 10 microU/mL insulin did not affect ET-1 production in the EC culture medium, a higher concentration of insulin stimulated it. Production of ET-1 in EC was activated by insulin via the IGF-1 receptor, inasmuch as alphaIR(3) blocked insulin mediated upregulation of ET-1. There was no significant difference in (3)H-thymidine incorporation in the presence of insulin (up to 1000 microU/mL) or TAK044. Culture medium from EC stimulated with insulin enhanced VSMC proliferation, which was almost totally suppressed by TAK044. Insulin induced VSMC growth dose dependently when VSMC were cultured alone. In contrast, insulin at concentrations of 100 microU/mL or lower failed to stimulate growth of co-cultured VSMC, but only at 330 microU/mL or higher concentrations stimulated VSMC growth in this system. Of interest, VSMC proliferation was greatest when L-NAME was added and co-cultured with EC. In summary, a severely hyperinsulinemic state may regulate VSMC and EC proliferation via activation of vasoactive substances such as ET-1 and nitric oxide induced by insulin.

Upregulation of cAMP is a new functional signal pathway of Klotho in endothelial cells.

We measured angiotensin I-converting enzyme (ACE) activity in a human endothelial cell to characterize the intracellular signal pathways of Klotho. COS-1 cells transfected with naked mouse membrane-form klotho plasmid DNA (pCAGGS-klotho) translated proper Klotho protein. This translated Klotho protein was secreted into the culture medium. Furthermore, ACE activity in human umbilical vein endothelial cells (HUVEC) was upregulated when HUVEC were co-cultured with COS-1 cells that were pre-transfected with pCAGGS-klotho. The conditioned medium from COS-1 cells pre-transfected with pCAGGS-klotho also dose-dependently upregulated ACE in HUVEC. In addition, the conditioned medium induced time- and dose-dependent enhancement of cAMP production in HUVEC. Rp-cAMP, an inhibitor of cAMP-dependent protein kinase A (PKA), inhibited the upregulation of ACE by Klotho protein. Our results suggest that mouse membrane-form Klotho protein acts as a humoral factor to increase ACE activity in HUVEC via a cAMP-PKA-dependent pathway. These findings may provide a new insight into the mechanism of Klotho protein.