Tissue-specific expression of senescence biomarkers in spontaneously hypertensive rats: evidence of premature aging in hypertension.

Background: Cellular senescence is an age-related physiological process that contributes to tissue dysfunction and accelerated onset of chronic metabolic diseases including hypertension. Indeed, elevation of blood pressure in hypertension coincides with premature vascular aging and dysfunction. In addition, onsets of metabolic disturbance and osteopenia in patients with hypertension have also been reported. It is possible that hypertension enhances premature aging and causes progressive loss of function in multiple organs. However, the landscape of cellular senescence in critical tissues affected by hypertension remains elusive. Materials and Methods: Heart, liver, bone, hypothalamus, and kidney were collected from spontaneously hypertensive rats (SHR) and age- and sex-matched normotensive Wistar rats (WT) at 6, 12, 24 and 36 weeks of age (n = 10 animals/group). Changes in mRNA levels of senescence biomarkers namely cyclin-dependent kinase (CDK) inhibitors (CDKIs), i.e., Cdkn2a (encoding p16Ink4a) and Cdkn1a (encoding p21cip1) as well as senescence-associated secretory phenotypes (SASPs), i.e., Timp1, Mmp12, Il6 and Cxcl1, were determined. Additionally, bone collagen alignment and hydroxy apatite crystal dimensions were determined by synchrotron radiation small- and wide-angle X-ray scattering (SAXS/WAXS) techniques. Results: Real-time PCR revealed that transcript levels of genes encoding CDKIs and SASPs in the heart and liver were upregulated in SHR from 6 to 36 weeks of age. Expression of Timp1 and Cxcl1 was increased in bone tissues isolated from 36-week-old SHR. In contrast, we found that expression levels of Timp1 and Il6 mRNA were decreased in hypothalamus and kidney of SHR in all age groups. Simultaneous SAXS/WAXS analysis also revealed misalignment of bone collagen fibers in SHR as compared to WT. Conclusion: Premature aging was identified in an organ directly affected by high blood pressure (i.e., heart) and those with known functional defects in SHR (i.e., liver and bone). Cellular senescence was not evident in organs with autoregulation of blood pressure (i.e., brain and kidney). Our study suggested that cellular senescence is induced by persistently elevated blood pressure and in part, leading to organ dysfunction. Therefore, interventions that can both lower blood pressure and prevent cellular senescence should provide therapeutic benefits for treatment of cardiovascular and metabolic consequences.

Excessive salt consumption causes systemic calcium mishandling and worsens microarchitecture and strength of long bones in rats.

Excessive salt intake has been associated with the development of non-communicable diseases, including hypertension with several cardiovascular consequences. Although the detrimental effects of high salt on the skeleton have been reported, longitudinal assessment of calcium balance together with changes in bone microarchitecture and strength under salt loading has not been fully demonstrated. To address these unanswered issues, male Sprague-Dawley rats were fed normal salt diet (NSD; 0.8% NaCl) or high salt diet (HSD; 8% NaCl) for 5 months. Elevation of blood pressure, cardiac hypertrophy and glomerular deterioration were observed in HSD, thus validating the model. The balance studies were performed to monitor calcium input and output upon HSD challenge. The HSD-induced increase in calcium losses in urine and feces together with reduced fractional calcium absorption led to a decrease in calcium retention. With these calcium imbalances, we therefore examined microstructural changes of long bones of the hind limbs. Using the synchrotron radiation x-ray tomographic microscopy, we showed that trabecular structure of tibia and femur of HSD displayed a marked increase in porosity. Consistently, the volumetric micro-computed tomography also demonstrated a significant decrease in trabecular bone mineral density with expansion of endosteal perimeter in the tibia. Interestingly, bone histomorphometric analyses indicated that salt loading caused an increase in osteoclast number together with decreases in osteoblast number and osteoid volume. This uncoupling process of bone remodeling in HSD might underlie an accelerated bone loss and bone structural changes. In conclusion, long-term excessive salt consumption leads to impairment of skeletal mass and integrity possibly through negative calcium balance.

Impairment of bone microstructure and upregulation of osteoclastogenic markers in spontaneously hypertensive rats.

Hypertension and osteoporosis are the major non-communicable diseases in the elderly worldwide. Although clinical studies reported that hypertensive patients experienced significant bone loss and likelihood of fracture, the causal relationship between hypertension and osteoporosis has been elusive due to other confounding factors associated with these diseases. In this study, spontaneously hypertensive rats (SHR) were used to address this relationship and further explored the biophysical properties and the underlying mechanisms. Long bones of the hind limbs from 18-week-old female SHR were subjected to determination of bone mineral density (BMD) and their mechanical properties. Using synchrotron radiation X-ray tomographic microscopy (SRXTM), femoral heads of SHR displayed marked increase in porosity within trabecular area together with decrease in cortical thickness. The volumetric micro-computed tomography also demonstrated significant decreases in trabecular BMD, cortical thickness and total cross-sectional area of the long bones. These changes also led to susceptibility of the long bones to fracture indicated by marked decreases in yield load, stiffness and maximum load using three-point bending tests. At the cellular mechanism, an increase in the expression of osteoclastogenic markers with decrease in the expression of alkaline phosphatase was found in primary osteoblast-enriched cultures isolated from long bones of these SHR suggesting an imbalance in bone remodeling. Taken together, defective bone mass and strength in hypertensive rats were likely due to excessive bone resorption. Development of novel therapeutic interventions that concomitantly target hypertension and osteoporosis should be helpful in reduction of unwanted outcomes, such as bone fractures, in elderly patients.

Anomalous bone changes in ovariectomized type 2 diabetic rats: inappropriately low bone turnover with bone loss in an estrogen-deficient condition.

Estrogen deprivation accelerates bone resorption, leading to imbalance of bone remodeling and osteoporosis in postmenopausal women. In the elderly, type 2 diabetes mellitus (T2DM) frequently coexists as an independent factor of bone loss. However, little is known about the skeletal changes in a combined condition of estrogen deficiency and T2DM. Herein, we performed ovariectomy (OVX) in nonobese Goto-Kakizaki (GK) T2DM rats to examine changes associated with calcium and phosphate metabolism and bone microstructures and strength. As expected, wild-type (WT) rats subjected to ovariectomy (OVX-WT) had low trabecular bone volume and serum calcium with increased dynamic histomorphometric and serum bone markers, consistent with the high turnover state. T2DM in GK rats also led to low trabecular volume and serum calcium. However, the dynamic histomorphometric markers of bone remodeling were unaffected in these GK rats, indicating the distinct mechanism of T2DM-induced bone loss. Interestingly, OVX-GK rats were found to have anomalous and unique changes in bone turnover-related parameters, i.e., decreased osteoblast and osteoclast surfaces with lower COOH-terminal telopeptide of type I collagen levels compared with OVX-WT rats. Furthermore, the levels of calciotropic hormones, i.e., parathyroid hormone and 1,25(OH)2D3, were significantly decreased in OVX-GK rats. Although the OVX-induced bone loss did not further worsen in GK rats, a three-point bending test indicated that OVX-GK bones exhibited a decrease in bone elasticity. In conclusion, T2DM and estrogen deficiency both led to microstructural bone loss, the appearance of which did not differ from each factor alone. Nevertheless, the combination worsened the integrity and suppressed the turnover, which might eventually result in adynamic bone disease.

Insulin does not rescue cortical and trabecular bone loss in type 2 diabetic Goto-Kakizaki rats.

In type 2 diabetes mellitus (T2DM), the decreased bone strength is often associated with hyperglycemia and bone cell insulin resistance. Since T2DM is increasingly reported in young adults, it is not known whether the effect of T2DM on bone would be different in young adolescents and aging adults. Here, we found shorter femoral and tibial lengths in 7-month, but not 13-month, Goto-Kakizaki (GK) T2DM rats as compared to wild-type rats. Bone µCT analysis showed long-lasting impairment of both cortical and trabecular bones in GK rats. Although insulin treatment effectively improved hyperglycemia, it was not able to rescue trabecular BMD and cortical thickness in young adult GK rats. In conclusion, insulin treatment and alleviation of hyperglycemia did not increase BMD of osteopenic GK rats. It is likely that early prevention of insulin resistance should prevail over treatment of full-blown T2DM-related osteopathy.

Pueraria mirifica extract and puerarin enhance proliferation and expression of alkaline phosphatase and type I collagen in primary baboon osteoblasts.

Phytoestrogen-rich Pueraria mirifica (PM) tuberous extract is a promising candidate for the development of anti-osteoporosis drugs for postmenopausal women, but its action has never been validated in humans or in non-human primates, which are more closely related to humans than rodents. In vitro study of non-human primate osteoblasts is thus fundamental to prepare for in vivo studies of phytoestrogen effects on primate bone. This study aimed to establish a culture system of baboon primary osteoblasts and to investigate the effects of PM extract and its phytoestrogens on these cells. Primary osteoblasts from adult baboon fibulae exhibited osteoblast characteristics in regard to proliferation, differentiation, mineralization, and estrogen receptor expression. They responded to 17ß-estradiol by increased proliferation rate and mRNA levels of alkaline phosphatase (ALP), type I collagen, and osteocalcin. After being exposed for 48 h to 100 µg/ml PM extract, 1000 nM genistein, or 1000 nM puerarin, primary baboon osteoblasts markedly increased the rate of proliferation and mRNA levels of ALP and type I collagen without changes in Runx2, osterix, or osteocalcin expression. PM extract, genistein, and puerarin also decreased the RANKL/OPG ratio, suggesting that they could decrease osteoclast-mediated bone resorption. However, neither PM extract nor its phytoestrogens altered calcium deposition in osteoblast culture. In conclusion, we have established baboon primary osteoblast culture, which is a new tool for bone research and drug discovery. Furthermore, the present results provide substantial support for the potential of PM extract and its phytoestrogens to be developed as therapeutic agents against bone fragility.

High dietary cholesterol masks type 2 diabetes-induced osteopenia and changes in bone microstructure in rats.

Type 2 diabetes mellitus (T2DM) often occurs concurrently with high blood cholesterol or dyslipidemia. Although T2DM has been hypothesized to impair bone microstructure, several investigations showed that, when compared to age-matched healthy individuals, T2DM patients had normal or relatively high bone mineral density (BMD). Since cholesterol and lipids profoundly affect the function of osteoblasts and osteoclasts, it might be cholesterol that obscured the changes in BMD and bone microstructure in T2DM. The present study, therefore, aimed to determine bone elongation, epiphyseal histology, and bone microstructure in non-obese T2DM Goto-Kakizaki rats treated with normal (GK-ND) and high cholesterol diet. We found that volumetric BMD was lower in GK-ND rats than the age-matched wild-type controls. In histomorphometric study of tibial metaphysis, T2DM evidently suppressed osteoblast function as indicated by decreases in osteoblast surface, mineral apposition rate, and bone formation rate in GK-ND rats. Meanwhile, the osteoclast surface and eroded surface were increased in GK-ND rats, thus suggesting an activation of bone resorption. T2DM also impaired bone elongation, presumably by retaining the chondrogenic precursor cells in the epiphyseal resting zone. Interestingly, several bone changes in GK rats (e.g., increased osteoclast surface) disappeared after high cholesterol treatment as compared to wild-type rats fed high cholesterol diet. In conclusion, high cholesterol diet was capable of masking the T2DM-induced osteopenia and changes in several histomorphometric parameters that indicated bone microstructural defect. Cholesterol thus explained, in part, why a decrease in BMD was not observed in T2DM, and hence delayed diagnosis of the T2DM-associated bone disease.

Defective bone microstructure in hydronephrotic mice: a histomorphometric study in ICR/Mlac-hydro mice.

Chronic renal impairment can lead to bone deterioration and abnormal bone morphology, but whether hydronephrosis is associated with bone loss remains unclear. Herein, we aimed to use computer-assisted bone histomorphometric technique to investigate microstructural bone changes in Imprinting Control Region (ICR) mice with a spontaneous mutation that was associated with bilateral nonobstructive hydronephrosis (ICR/Mlac-hydro). The results showed that 8-week-old ICR/Mlac-hydro mice manifested decreases in trabecular bone number and thickness, and an increased trabecular separation, thereby leading to a reduction in trabecular bone volume compared with the wild-type mice. Furthermore, histomorphometric parameters related to both bone resorption and formation, that is, eroded surface, osteoclast surface, and osteoblast surface, were much lower in ICR/Mlac-hydro mice than in the wild type. A decrease in moment of inertia was found in ICR/Mlac-hydro mice, indicating a decrease in bone strength. In conclusion, ICR/Mlac-hydro mice exhibited trabecular bone loss, presumably caused by marked decreases in both osteoblast and osteoclast activities, which together reflected abnormally low bone turnover. Thus, this mouse strain appeared to be a valuable model for studying the hydronephrosis-associated bone disease.

Upregulation of osteoblastic differentiation marker mRNA expression in osteoblast-like UMR106 cells by puerarin and phytoestrogens from Pueraria mirifica.

Phytoestrogens have attracted attention for their potential in the prevention of postmenopausal osteoporosis. Recently, phytoestrogen-rich herb Pueraria mirifica has been demonstrated to possess an osteogenic effect on bone in ovariectomized rats, but its underlying cellular mechanism was not known. Here, we investigated the effects of P. mirifica extract and its major isoflavone compound, puerarin, on cell viability, cell proliferation and the expression of differentiation markers in rat osteoblast-like UMR106 cells. After exposure to 17ß-estradiol (E2), genistein, P. mirifica extract and puerarin, proliferation but not viability of UMR106 cells was markedly decreased. Quantitative real-time PCR revealed that P. mirifica extract and puerarin significantly increased the mRNA expression of alkaline phosphatase (ALP) and osteoprotegerin, but not Runx2, osterix or osteocalcin. Puerarin also decreased the mRNA expression of receptor activator of nuclear factor-κB ligand, an osteoclastogenic factor, suggesting that it could induce bone gain by enhancing osteoblast differentiation and suppressing osteoclast function. Furthermore, after an exposure to high affinity estrogen receptor (ER) antagonist (ICI182780), the E2-, genistein-, P. mirifica extract- and puerarin-induced upregulation of ALP expressions were completely abolished. It could be concluded that P. mirifica extract and puerarin induced osteoblast differentiation rather than osteoblast proliferation in an ER-dependent manner. The present findings, therefore, corroborated the potential benefit of P. mirifica extract and puerarin in the prevention and treatment of postmenopausal osteoporosis.