Assessment of the synergic effect of immunomodulation on nerve repair using multiparametric magnetic resonance imaging.

INTRODUCTION: The immune system plays a pivotal role in nerve injury. The aim of this study was to determine the role of multiparametric magnetic resonance imaging (MRI) in evaluation of the synergic effect of immunomodulation on nerve regeneration in neurotmesis. METHODS: Rats with sciatic nerve neurotmesis and surgical repair underwent serial multiparametric MR examinations over an 8-week period after subepineurial microinjection of lipopolysaccharide (LPS) and subsequent subcutaneous injection of FK506 or subepineurial microinjection of LPS or phosphate-buffered saline (PBS) alone. RESULTS: Nerves treated with immunomodulation showed more prominent regeneration than those treated with LPS or PBS alone and more rapid restoration toward normal T2, fractional anisotropy (FA), and radial diffusivity (RD) values than nerves injected with LPS or PBS. DISCUSSION: Nerves treated with immunomodulation exert synergic beneficial effects on nerve regeneration that can be predicted by T2 measurements and FA and RD values. Muscle Nerve 57: E38-E45, 2018.

Monitoring of macrophage recruitment enhanced by Toll-like receptor 4 activation with MR imaging in nerve injury.

INTRODUCTION: Macrophage recruitment is critical for nerve regeneration after an injury. The aim of this study was to investigate whether ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle-based MRI could be used to monitor the enhanced macrophage recruitment by Toll-like receptor 4 (TLR4) activation in nerve injury. METHODS: Rats received intraperitoneal injections of either lipopolysaccharide (LPS) or phosphate buffered saline (PBS) or no injection (controls) after a sciatic nerve crush injury. After intravenous injection of the USPIOs (LPS and PBS groups) or PBS (control group), MRI was performed and correlated with histological findings. RESULTS: LPS group showed more remarkable hypointense signals on T2*-weighted imaging and lower T2 values in the crushed nerves than PBS group. The hypointense signal areas were associated with an enhanced recruitment of iron-loaded macrophages to the injured nerves. DISCUSSION: USPIO-enhanced MRI can be used to monitor the enhanced macrophage recruitment by means of TLR4 signal pathway activation in nerve injury. Muscle Nerve, 2018.

DTI metrics can be used as biomarkers to determine the therapeutic effect of stem cells in acute peripheral nerve injury.

PURPOSE: To determine the role of diffusion tensor imaging (DTI) metrics as biomarkers for the therapeutic effects of mesenchymal stem cells (MSCs) in acute peripheral nerve injury. MATERIALS AND METHODS: Forty-four adult rats received subepineurial microinjection of MSCs (n = 22) or phosphate buffered saline (PBS, n = 22) 1 week after the sciatic nerve trunk crush injury. Sequential fat-suppressed T2-weighted imaging, T2 measurement, DTI and sciatic nerve functional assessment were performed at a 3.0 Tesla MR unit over an 8-week follow-up, with histological assessments performed at regular intervals. The sciatic nerve function index, T2 value, and DTI metrics, including fractional anisotropy (FA), axial diffusivity, radial diffusivity (RD), and mean diffusivity values of the distal stumps of crushed nerves were measured and compared between the two groups. RESULTS: Nerves treated with MSCs showed better functional recovery and exhibited more pronounced nerve regeneration compared with nerves treated with PBS. T2 values in nerves treated with MSCs or PBS showed a similar change pattern (P = 0.174), while FA and RD values in nerves treated with MSCs showed more rapid return (one week earlier) to baseline level than nerves treated with PBS (P = 0.045; 0.035). Nerves treated with MSCs had higher FA and lower RD values than nerves treated with PBS during the period from 2 to 3 weeks after surgery (P ≤ 0.0001, 0.004; P = 0.004, 0.006). CONCLUSION: FA and RD values derived from DTI might be used as sensitive biomarkers for detecting the therapeutic effect of stem cells in acute peripheral nerve crush injuries. LEVEL OF EVIDENCE: 2 J. Magn. Reson. Imaging 2017;45:855-862.

Directly Insight Into the Inter- and Intramolecular Interactions of CL-20/TNT Energetic Cocrystal through the Theoretical Simulations of THz Spectroscopy.

Compared with cocrystal coformers, an explosive cocrystal has distinctive packing arrangements and complex intermolecular interactions. Identifying the spectral signatures of an explosive cocrystal and understanding the molecular low-frequency modes by means of the spectrum in the terahertz range are of great worth to the explicit mechanism of cocrystal formation. In this work, on the basis of the joint molecular dynamics (MD) simulations and solid-state density functional theory (DFT) calculations, we have investigated the terahertz (THz) absorption spectra of the CL-20/TNT cocrystal and its different directions as well as cocrystal coformers and determined the systematic and all-sided assignments of corresponding THz vibration modes. The THz spectral comparison of the cocrystal with different directions and the cocrystal coformers indicates that the CL-20/TNT cocrystal has five fresh low-frequency absorption features as unique and discernible peaks for identification, in which 0.25, 0.73, and 0.87 THz are attributed to intensive crystalline vibrations; 0.87 THz is also caused by C-H···O hydrogen-bonding bending vibrations; 1.60 and 1.85 THz features originate from C-H···O hydrogen-bond stretching vibrations. Additionally, the THz spectrum of the (001) direction of the CL-20/TNT cocrystal verifies that the molecular conformation of the CL-20 is the same as that in the ß-polymorph, other than the initial conformation of raw material ε-CL-20.

Thyroid hormone attenuates vascular calcification induced by vitamin D3 plus nicotine in rats.

Thyroid hormones (THs) including thyroxine (T4) and triiodothyronine (T3) play critical roles in bone remodeling. However, the role and mechanism of THs in vascular calcification (VC) have been unclear. To explore the pathophysiological roles of T3 on VC, we investigated the changes in plasma and aortas of THs concentrations and the effect of T3 on rat VC induced by vitamin D3 plus nicotine (VDN). VDN-treated rat showed decreased plasma T3 content, increased vascular calcium deposition, and alkaline phosphatase (ALP) activity. Administration of T3 (0.2 mg/kg body weight IP) for 10 days greatly reduced vascular calcium deposition and ALP activity in calcified rat aortas when compared with controls. Concurrently, the loss of smooth muscle lineage markers α-actin and SM22a was restored, and the increased bone-associated molecules, such as runt-related transcription factor2 (Runx2), Osterix, and osteopontin (OPN) levels in calcified aorta, were reduced by administration of T3. The suppression of klotho in calcified rat aorta was restored by T3. Methimazole (400 mg/L) blocked the beneficial effect of T3 on VC. These results suggested that T3 can inhibit VC development.

Activating transcription factor 4 is involved in endoplasmic reticulum stress-mediated apoptosis contributing to vascular calcification.

Our previous work reported that endoplasmic reticulum stress (ERS)-mediated apoptosis was activated during vascular calcification (VC). Activating transcription factor 4 (ATF4) is a critical transcription factor in osteoblastogenesis and ERS-induced apoptosis. However, whether ATF4 is involved in ERS-mediated apoptosis contributing to VC remains unclear. In the present study, in vivo VC was induced in rats by administering vitamin D3 plus nicotine. Vascular smooth muscle cell (VSMC) calcification in vitro was induced by incubation in calcifying media containing ß-glycerophosphate and CaCl2. ERS inhibitors taurine or 4-phenylbutyric acid attenuated ERS and VSMC apoptosis in calcified rat arteries, reduced calcification and retarded the VSMC contractile phenotype transforming into an osteoblast-like phenotype in vivo. Inhibition of ERS retarded the VSMC phenotypic transition into an osteoblast-like cell phenotype and reduced VSMC calcification and apoptosis in vitro. Interestingly, ATF4 was activated in calcified aortas and calcified VSMCs in vitro. ATF4 knockdown attenuated ERS-induced apoptosis in calcified VSMCs. ATF4 deficiency blocked VSMC calcification and negatively regulated the osteoblast phenotypic transition of VSMCs in vitro. Our results demonstrate that ATF4 was involved at least in part in the process of ERS-mediated apoptosis contributing to VC.

[Genetics and clinical study of Chinese kindreds with dentatorubral pallidoluysian atrophy].

OBJECTIVE: To investigate genetics and clinical characteristics of dentatorubral-pallidoluysian atrophy (DRPLA) in Chinese kindreds. METHODS: Fragment analysis with laser-induced fluorescence in capillary electrophoresis was performed for the cytosine-adenine-guanine (CAG) repeats of DRPLA gene in 708 probands of autosomal dominant ataxia pedigrees and 119 sporadic ataxia cases. RESULTS: Expanded CAG repeats of DRPLA gene were detected in probands of three ataxia pedigrees, with the numbers of repeats being 16/58, 16/58 and 14/54, respectively. In addition to ataxia, patients with adult-onset disease also exhibited spasm and neck torsion. CONCLUSION: Only three cases of DRPLA have been identified among 827 cases, which suggested that DRPLA is a relatively rare subtype of SCA in Chinese population. Clinical variation among the patients suggested DRPLA has a wide spectrum of phenotype.

[Effect of Baicalin on Pyroptosis of Diffuse Large B-Cell Lymphoma Cell Lines DB and Its Mechanism].

OBJECTIVE: To investigate the effect of Baicalin on the proliferation and pyroptosis of diffuse large B-cell lymphoma cell line DB and its mechanism. METHODS: DB cells were treated with baicalin at different concentrations (0, 5, 10, 20, 40 µmol/L). Cell proliferation was detected by CCK-8 assay and half maximal inhibitory concentration (IC50) was calculated. The morphology of pyroptosis was observed under an inverted microscope, the integrity of the cell membrane was verified by LDH content release assay, and the expressions of pyroptosis-related mRNA and protein (NLRP3, GSDMD, GSDME, N-GSDMD, N-GSDME) were detected by real-time fluorescence quantitative PCR and Western blot. In order to further clarify the relationship between baicalin-induced pyroptosis and ROS production in DB cells, DB cells were divided into control group, baicalin group, NAC group and NAC combined with baicalin group. DB cells in the NAC group were pretreated with ROS inhibitor N-acetylcysteine (NAC) 2 mmol/L for 2 h. Baicalin was added to the combined treatment group after pretreatment, and the content of reactive oxygen species (ROS) in the cells was detected by DCFH-DA method after 48 hours of culture. RESULTS: Baicalin inhibited the proliferation of DB cells in a dose-dependent manner (r=-0.99), and the IC50 was 20.56 µmol/L at 48 h. The morphological changes of pyroptosis in DB cells were observed under inverted microscope. Compared with the control group, the release of LDH in the baicalin group was significantly increased (P<0.01), indicating the loss of cell membrane integrity. Baicalin dose-dependently increased the expression levels of NLRP3, N-GSDMD, and N-GSDME mRNA and protein in the pyroptosis pathway (P<0.05). Compared with the control group, the level of ROS in the baicalin group was significantly increased (P<0.05), and the content of ROS in the NAC group was significantly decreased (P<0.05). Compared with the NAC group, the content of ROS in the NAC + baicalin group was increased. Baicalin significantly attenuated the inhibitory effect of NAC on ROS production (P<0.05). Similarly, Western blot results showed that compared with the control group, the expression levels of pyroptosis-related proteins was increased in the baicalin group (P<0.05). NAC inhibited the expression of NLRP3 and reduced the cleavage of N-GSDMD and N-GSDME (P<0.05). Compared with the NAC group, the NAC + baicalin group had significantly increased expression of pyroptosis-related proteins. These results indicate that baicalin can effectively induce pyroptosis in DB cells and reverse the inhibitory effect of NAC on ROS production. CONCLUSION: Baicalin can inhibit the proliferation of DLBCL cell line DB, and its mechanism may be through regulating ROS production to affect the pyroptosis pathway.

Radio- and Photosensitizing Os(II)-Based Nanocage for Combined Radio-/Chemo-/X-ray-Induced Photodynamic Therapies, NIR Imaging, and Drug Delivery.

Integration of clinical imaging and collaborative multimodal therapies into a single nanomaterial for multipurpose diagnosis and treatment is of great interest to theranostic nanomedicine. Here, we report a rational design of a discrete Os-based metal-organic nanocage Pd6(OsL3)828+ (MOC-43) as a versatile theranostic nanoplatform to meet the following demands simultaneously: (1) synergistic treatments of radio-, chemo-, and X-ray-induced photodynamic therapies (X-PDT) for breast cancer, (2) NIR imaging for cancer cell tracking and tumor-targeting, and (3) anticancer drug transport through a host-guest strategy. The nanoscale MOC-43 incorporates high-Z Os-element to interact with X-ray irradiation for dual radiosensitization and photosensitization, showing efficient energy transfer to endogenous oxygen in cancer cells to enhance X-PDT efficacy. It also features intrinsic NIR emission originating from metal-to-ligand charge transfer (MLCT) as an excellent imaging probe. Meanwhile, its 12 pockets can capture and concentrate low-water-soluble molecules for anticancer drug delivery. These multifunctions are implemented and demonstrated by micellization of coumarin-loaded cages with DSPE-PEG2000 into coumarin ⊂ MOC-43 nanoparticles (CMNPs) for efficient subcellular endocytosis and uptake. The cancer treatments in vitro/in vivo show promising antitumor performance, providing a conceptual protocol to combine cage-cargo drug transport with diagnosis and treatment for collaborative cancer theranostics by virtue of multifunction synergism on a single-nanomaterial platform.

Effectiveness of lenvatinib plus immune checkpoint inhibitors in primary advanced hepatocellular carcinoma beyond oligometastasis.

BACKGROUND: Targeted therapy combined with immune checkpoint inhibitors is considered a promising treatment for primary advanced hepatocellular carcinoma (HCC). Nevertheless, the difference between synchronous and asynchronous treatment of lenvatinib with programmed death receptor-1 (PD-1) inhibitor in advanced HCC is still unclear. The aim of this investigation is to evaluate the effectiveness of synchronous and asynchronous of lenvatinib and PD-1 inhibitor on the advanced HCC beyond oligometastasis. METHODS: In this study, 213 patients from four institutions in China were involved. Patients were split into two collections: (1) lenvatinib plus PD-1 inhibitor were used synchronously (synchronous treatment group); (2) patients in asynchronous treatment group received PD-1 inhibitor after 3 months of lenvatinib treatment prior to tumour progression. To analyse progression-free survival (PFS), overall survival (OS), efficacy and safety of patients in both groups, we employed propensity score matching (PSM). RESULTS: The 6-, 12- and 24-month OS rates were 100%, 93.4% and 58.1% in the synchronous treatment group and 100%, 71.5% and 25.3% in the asynchronous treatment group, respectively. In contrast to the asynchronous treatment group, the group treated synchronously exhibited a substantially enhanced OS (hazard ratio [HR], 0.45; 95% confidence interval [CI], 0.30-0.66; p < .001). The 6-, 12- and 18-month PFS rates were 82.6%, 42.6% and 10.8% in the synchronous treatment group and 63.3%, 14.2% and 0% in the asynchronous treatment group, respectively. A significant difference was observed in the PFS rate (HR, 0.46; 95% CI, 0.33-0.63; p < .001) between the two collections. CONCLUSIONS: Patients with advanced HCC beyond oligometastasis, simultaneous administration of lenvatinib and PD-1 inhibitor led to significant improvements in survival.

[Regulation of Baicalin on Growth of Extranodal NK/T Cell Lymphoma Cells through FOXO3/CCL22 Signaling Pathway].

OBJECTIVE: To investigate the effect of baicalin on the growth of extranodal NK/T cell lymphoma (ENKTCL) cells and its related mechanism. METHODS: Normal NK cells and human ENKTCL cells lines SNK-6 and YTS were cultured, then SNK-6 and YTS cells were treated with 5, 10, 20 µmol/L baicalin and set control. Cell proliferation and apoptosis was detected by Edu method and FCM method, respectively, and expressions of BCL-2, Bax, FOXO3 and CCL22 proteins were detected by Western blot. Interference plasmids were designed and synthesized. FOXO3 siRNA interference plasmids and CCL22 pcDNA overexpression plasmids were transfected with PEI transfection reagent. Furthermore, animal models were established for validation. RESULTS: In control group and 5, 10, 20 µmol/L baicalin group, the proliferation rate of SNK-6 cells was (56.17±2.96)%, (51.92±4.63)%, (36.42±1.58)%, and (14.60±2.81)%, respectively, while that of YTS cells was (58.85±2.98)%, (51.38±1.32)%, (34.75±1.09)%, and (15.45±1.10)%, respectively. In control group and 5, 10, 20 µmol/L baicalin group, the apoptosis rate of SNK-6 cells was (5.93±0.74)%, (11.78±0.34)%, (28.46±0.44)%, and (32.40±0.37)%, respectively, while that of YTS cells was (7.93±0.69)%, (16.29±1.35)%, (33.91±1.56)%, and (36.27±1.06)%, respectively. Compared with control group, the expression of BCL-2 protein both in SNK-6 and YTS cells decreased significantly (P<0.001), and the expression of Bax protein increased in SNK-6 cells only when the concentration of baicalin was 20 µmol/L (P<0.001), while that in YTS cells increased in all three concentrations(5, 10, 20 µmol/L) of baicalin (P<0.001). The expression of FOXO3 protein decreased while CCL22 protein increased in ENKTCL cell lines compared with human NK cells (P<0.001), but the expression of FOXO3 protein increased (P<0.01) and CCL22 protein decreased after baicalin treatment (P<0.001). Animal experiments showed that baicalin treatment could inhibit tumor growth. The expression of CCL22 protein in ENKTCL tissue of nude mice treated with baicalin decreased compared with control group (P<0.01), while the FOXO3 protein increased (P<0.05). In addition, FOXO3 silencing resulted in the decrease of FOXO3 protein expression and increase of CCL22 protein expression (P<0.01, P<0.001). CONCLUSION: Baicalin can inhibit proliferation and promote apoptosis of ENKTCL cell lines SNK-6 and YTS, up-regulate the expression of Bax protein, down-regulate the expression of BCL-2 protein, and down-regulate the expression of CCL22 protein mediated by FOXO3. Animal experiment shown that the baicalin can inhibit tumor growth. Baicalin can inhibit the growth and induce apoptosis of ENKTCL cells through FOXO3/CCL22 signaling pathway.

Suppression of experimental abdominal aortic aneurysm in a rat model by the phosphodiesterase 3 inhibitor cilostazol.

BACKGROUND: The present experiments sought to determine whether cilostazol, a selective inhibitor of cyclic adenosine monophosphate (cAMP) phosphodiesterase 3 (PDE3), suppressed elastase-induced abdominal aortic aneurysm (AAA) development in a rat model. METHODS: Male Sprague-Dawley rats (n = 16/each group) were randomly distributed into three groups: sham-, saline-, and cilostazol-. Rats of saline and cilostazol groups underwent intra-aortic elastase perfusion to induce AAAs, while rats of sham-group were perfused with saline. Rats of cilostazol-group received cilostazol treatment (100 mgkg(-1)d(-1)) for the entire experimental period. The areas of the lumen of the aortas at the segment with maximum diameter were measured preperfusion and on d 7, 14 after perfusion. Systolic blood pressure was measured by tail-cuff technique. Aortic tissue samples were harvested on d 14 after intra-aortic perfusion and evaluated by reverse transcription-polymerase chain reaction and Western blot for matrix metalloproteinase-2, 9 (MMP-2, 9), by immunohistochemistry for nuclear factor kappa B (NF-κB), and by Gomori aldehyde fuchsin for elastin. Activity of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and level of reactive oxygen species (ROS) in these samples were also measured. RESULTS: On d 14, rats of saline-group had significantly increased aortic sizes compared with sham-group (P < 0.01), while, cilostazol treatment significantly reduced this increase (cilostazol- versus saline-, P < 0.01) without affecting blood pressure (P > 0.05). The expression of both MMP-2 and MMP-9 and the destruction of elastic fibers in aortic tissues were significantly decreased by cilostazol treatment (P < 0.05), probably through the suppression of NF-κB activation (P < 0.01). Consistently, cilostazol significantly inhibited NADPH oxidase activity (P < 0.01), accompanied by a reduced level of ROS (P < 0.01). CONCLUSION: Cilostazol retards experimental AAAs development independently of blood pressure reduction possibly by inhibiting proteolysis, inflammation, and oxidative stress. Selective PDE3 inhibition may offer an additional method to pharmacologically inhibit AAAs.

Transplanted neural stem cells promote nerve regeneration in acute peripheral nerve traction injury: assessment using MRI.

OBJECTIVE: The purpose of our study was to monitor neural stem cells (NSCs) transplanted in acute peripheral nerve traction injury and to use MRI to assess the ability of NSCs to promote nerve regeneration. MATERIALS AND METHODS: After labeling with gadolinium-diethylene triamine pentaacetic acid (gadopentetate dimeglumine) and fluorescent dye (PKH26), 5 × 10(5) NSCs were grafted to acutely distracted sciatic nerves in 21 New Zealand White rabbits. In addition, 5 × 10(5) unlabeled NSCs (n = 21) and vehicle alone (n = 21) subjects were injected as a control. Serial MRI was performed with a 1.5-T scanner to determine the distribution of grafted cells. Sequential T1 and T2 values of the nerves and functional recovery were measured over a 70-day follow-up period, with histologic assessments performed at regular intervals. RESULTS: The distribution and migration of labeled NSCs could be tracked with MRI until 10 days after transplantation. Compared with vehicle control, nerves grafted with labeled or unlabeled NSCs had better functional recovery and showed improved nerve regeneration but exhibited a sustained increase of T1 and T2 values during the phase of regeneration. CONCLUSION: Gadopentetate dimeglumine-based labeling allowed short-term in vivo MRI tracking of NSCs grafted in injured nerves. NSCs transplantation could promote nerve regeneration in acute peripheral nerve traction injury as shown by a prolonged increase of nerve T1 and T2 values.

MR neurography: T1 and T2 measurements in acute peripheral nerve traction injury in rabbits.

PURPOSE: To prospectively evaluate magnetic resonance (MR) signal abnormalities and the time course of T1 and T2 values in a rabbit model of acute nerve traction injury with histologic and functional recovery correlation. MATERIALS AND METHODS: All experimental protocols were approved by the institutional animal use and care committee. Acute traction injury was produced in the sciatic nerve of one hind limb in each of 28 rabbits. The contralateral sham-operated nerves served as controls. Sequential MR imaging and T1 and T2 measurements, as well as measurements of functional changes, were obtained over a 70-day follow-up period, with histologic assessments performed at regular intervals. Signal abnormalities and the time course of T1 and T2 values were observed in the proximal, traction, and distal portions of the injured nerves and the sham-operated nerves, and were compared with each other. RESULTS: Nerves with acute traction injury showed visible hyperintense signals on T2-weighted images and had prolonged T1 and T2 values. Differences of T1 and T2 values were dependent on the sites along the same injured nerve, with the most pronounced and prolonged phase of T1 and T2 increases (peak values of 1333 msec +/- 46 and 79 msec +/- 3.7, respectively) observed in the most severely damaged portion of the injured nerve. T1 and T2 values and functional changes after nerve injury showed a similar time course. A return of T1 and T2 signals to normal values correlated with functional improvement. CONCLUSION: MR imaging could be used to help predict the degree of nerve damage and monitor the process of nerve recovery in acute peripheral nerve traction injury. (c) RSNA, 2010.

In vivo MR imaging tracking of transplanted mesenchymal stem cells in a rabbit model of acute peripheral nerve traction injury.

PURPOSE: To investigate in vivo MRI tracking mesenchymal stem cells (MSCs) in peripheral nerve injures using a clinically available paramagnetic contrast agent (Gd-DTPA) and commercially available rhodamine-incorporated transfection reagents (PEI-FluoR). MATERIALS AND METHODS: After bone marrow MSCs were labeled with Gd-DTPA and PEI-FluoR complex, the labeling efficacy and longevity of Gd-DTPA maintenance were measured and cell viability, proliferation, and apoptosis were assessed. Thirty-six rabbits with acute sciatic nerve traction injury randomly received 1 × 10(6) labeled (n = 12) or unlabeled MSCs (n = 12) or vehicle alone injection. The distribution and migration of implanted cells was followed by MRI and correlated with histology. The relative signal intensity (RSL) of the grafts was measured. RESULTS: The labeling efficiency was 76 ± 4.7% and the labeling procedure did not influence cell viability, proliferation, and apoptosis. A persistent higher RSL in grafts was found in the labeled group compared with the unlabeled and vehicle groups until 10 days after transplantation (P < 0.05). The distribution and migration of labeled cells could be tracked by MRI until 10 days after transplantation. Transplanted MSCs were not found to transdifferentiate into Schwann-like cells within 14-day follow-up. CONCLUSION: Labeling MSCs with the dual agents may enable cellular MRI of the engraftment in the experimental peripheral nerve injury.

Adrenomedullin up-regulates osteopontin and attenuates vascular calcification via the cAMP/PKA signaling pathway.

AIM: To determine whether adrenomedullin (ADM) attenuates vascular calcification (VC) by inducing osteopontin (OPN) expression. METHODS: A VC model of rat aorta was induced with vitamin D3 plus nicotine (VDN), and vascular smooth muscle cell (VSMC) calcification was induced with beta-glycerophosphate. Von Kossa staining and alizarin red staining were assessed. Alkaline phosphatase (ALP) activity was measured. Immunohistochemical analysis was used to detect alpha-actin, while RT-PCR and Western blot analysis were used to quantify OPN expression. RESULTS: Administration of ADM greatly reduced VC in VDN-treated aortas compared with controls, which was confirmed in calcified VSMCs. The decrease in alpha-actin expression was ameliorated by ADM both in vivo and in vitro. Moreover, mRNA and protein expression levels of OPN were significantly up-regulated in calcified aortas, and ADM increased OPN expression in calcified aortas. Furthermore, ADM up-regulated OPN expression in normal aortas and VSMCs. The ADM-mediated effects were similar to that of forskolin, which activates adenylyl cyclase; additionally, while the PKA inhibitor H89 and Ca²(+) chelator Fura-2 blocked the effect of ADM. However, the MEK/ERK inhibitor PD98509 had no effect on ADM induction of OPN mRNA expression. An OPN polyclonal antibody inhibited ADM-mediated attenuation of VC. CONCLUSION: ADM up-regulates OPN expression and thus attenuates VC via PKA. ADM appears to be an endogenous cardiovascular protective peptide and may represent a new therapeutic target for VC treatment.

[Effect of age on vascular calcification induced by vitamin D3 and nicotine].

OBJECTIVE: To explore the effect of age on vascular calcification induced by vitamin D3 and nicotine. METHODS: Vascular calcification in rats was induced by administration of vitamin D3 plus nicotine (VDN treatment). After six weeks, Von Kossa staining, calcium content, alkaline phosphatase activity, phosphorus and calcium content in plasma were assayed. Carotid blood pressure, cardiac function and the relative amounts of osteopontin (OPN), osteoprotegerin (OPG), matrix Gla protein (MGP), bone morphogenetic protein-2 (BMP2) mRNA level and smooth muscle actin-alpha (alpha-SMA)protein level were measured. RESULTS: Compared with control group, the systolic blood pressure(SBP)of the rats of 2,8 and 16 months with vascular calcification respectively increased by 20.7%, 29.4% and 22.2% (P<0.05); the left ventricular systolic pressure (LVSP) respectively increased by 13.6%, 21.1% and 16.2% (P<0.05); + LVdP/dtmax respectively increased by 49.1% (P<0.01), 21.4% and 13.1% (P<0.05); -LVdP/dtmax respectively increased by 56.3% (P<0.01), 24.4% and 11.3% (P<0.05). Aortic calcium contents of the 2-, 8- and 16-month calcified rats were respectively 2.62-fold (P<0.05), 24.87-fold (P<0.01) and 10.01-fold (P<0.05) of the age-matched control group. As compared with the aortic calcium contents of calcified groups at different ages, the calcification group of 8 months had higher aortic calcium content than those of 2 and 16 months, which were respectively, 5.28-fold and 2.63-fold (P<0.05). Compared with the control groups, alkaline phosphatases activity (ALP) of calcification groups increased respectively by 126.6%, 115.2% and 227.9% (P<0.01) in the 2-, 8- and 16-month rats. As compared with the ALP activity of calcified groups at different ages, ALP activity of aortic calcification group of the 8-month-old rats was higher than that of the 2-month-old and 16-month-old rats, which increased by 176% and 75% respectively (all P<0.01). Von kossa staining for calcification showed positive staining as black/brown areas within the main, large, nodular structures as shown in extracellular matrix and cytoplasma in VDN groups at different ages, especially in the 8-month-old VDN group, with the most dispersed calcific nodules deposited and a few of the elastic fibers of the medial layer collapse. The mRNA expressions of OPN, OPG, MGP, BMP2 were up-regulated (P<0.01 or P<0.05) and protein levels of alpha-SMA were down-regulated in different calcification groups(P<0.05). The mRNA levels of OPN in 8-month-old calcification group increased by 3.41-fold (P<0.01) and 1.34-fold (P<0.05) respectively compared with the 2-month-old and 16-month-old calcification groups. And the alpha-SMA protein expression levels were lower at calcification groups in different ages, which were respectively equivalent to 17.6% of the 2-month-old control group (P<0.01), 11% of the 8-month-old control group (P<0.05) and 41.7% of 16-month-old control group (P<0.01). CONCLUSION: SD rats of 2, 8 and 16 months can all be used to duplicate vascular calcification model induced by vitamin D3 plus nicotine and the 8-month-old rat has the most sensitivity to the calcification treatment, which means that the 8-month-old rat may be the most appropriate age for the study of vascular calcification.

[The endocrine function of the skeleton].

Skeleton has long been recognized as a organ supporting body system and regulating metabolism of calcium and phosphorus. Recent researches found that bone cells, especially osteoblast and osteoclast, synthesize and secrete various bioactive molecules, such as bone morphogenetic proteins, growth factors, adipokines, inflammatory cytokines and cardiovascular bioactive peptides. The active factors produced by bone not only play important roles in the skeleton system per se by paracrine/autocrine pathway but also regulate energy metabolism, inflammatory process, endocrine homeostasis by endocrine pathway.