Granulocyte colony-stimulating factor attenuates myocardial remodeling and ventricular arrhythmia susceptibility via the JAK2-STAT3 pathway in a rabbit model of coronary microembolization.

BACKGROUND: Coronary microembolization (CME) has a poor prognosis, with ventricular arrhythmia being the most serious consequence. Understanding the underlying mechanisms could improve its management. We investigated the effects of granulocyte colony-stimulating factor (G-CSF) on connexin-43 (Cx43) expression and ventricular arrhythmia susceptibility after CME. METHODS: Forty male rabbits were randomized into four groups (n = 10 each): Sham, CME, G-CSF, and AG490 (a JAK2 selective inhibitor). Rabbits in the CME, G-CSF, and AG490 groups underwent left anterior descending (LAD) artery catheterization and CME. Animals in the G-CSF and AG490 groups received intraperitoneal injection of G-CSF and G-CSF + AG490, respectively. The ventricular structure was assessed by echocardiography. Ventricular electrical properties were analyzed using cardiac electrophysiology. The myocardial interstitial collagen content and morphologic characteristics were evaluated using Masson and hematoxylin-eosin staining, respectively. RESULTS: Western blot and immunohistochemistry were employed to analyze the expressions of Cx43, G-CSF receptor (G-CSFR), JAK2, and STAT3. The ventricular effective refractory period (VERP), VERP dispersion, and inducibility and lethality of ventricular tachycardia/fibrillation were lower in the G-CSF than in the CME group (P < 0.01), indicating less severe myocardial damage and arrhythmias. The G-CSF group showed higher phosphorylated-Cx43 expression (P < 0.01 vs. CME). Those G-CSF-induced changes were reversed by A490, indicating the involvement of JAK2. G-CSFR, phosphorylated-JAK2, and phosphorylated-STAT3 protein levels were higher in the G-CSF group than in the AG490 (P < 0.01) and Sham (P < 0.05) groups. CONCLUSION: G-CSF might attenuate myocardial remodeling via JAK2-STAT3 signaling and thereby reduce ventricular arrhythmia susceptibility after CME.

A retrospective study of 30,959 implants: Risk factors associated with early and late implant loss.

AIM: This retrospective study assessed the risk factors associated with early and late implant loss at the patient- and implant-based analysis. MATERIALS AND METHODS: A total of 18,199 patients received 30,959 dental implants during the years 2011-2015. Age, gender, jaw, location, implant brands, implant length and diameter, bone augmentation procedures, and the number of implants placed per patient were recorded. A multivariate generalized estimating equation (GEE) logistic regression was used to identify risk factors related to both early and late implant loss. RESULTS: The cumulative survival rates were 98.0% for patients and 98.7% for implants after 1-6 years observation time. A total of 183 patients with 194 implants were lost before or at the abutment connection, and 193 patients with 209 implants were lost after occlusal loading of the implant fixture. The multivariable GEE logistic regression showed that males, patients aged ≥41 years, and mandibular anterior location were risk factors for early implant loss. In the case of late implant loss, males, patients aged ≥41 years, bone augmentation and short implants were correlated with a significantly increased failure rate. CONCLUSIONS: General factors such as male sex, elderly patients, mandibular anterior location, bone augmentation and short implants were associated with implant loss.

Prostate Cancer Risk and DNA Methylation Signatures in Aging Rats following Developmental BPA Exposure: A Dose-Response Analysis.

BACKGROUND: Previous studies have uncovered heightened prostatic susceptibility to hormone-induced neoplasia from early-life exposure to low-dose bisphenol A (BPA). However, significant data gaps remain that are essential to address for biological relevance and necessary risk assessment. OBJECTIVES: A complete BPA dose-response analysis of prostate lesions across multiple prostatic lobes was conducted that included internal BPA dosimetry, progression to adenocarcinoma with aging and mechanistic connections to epigenetically reprogramed genes. METHODS: Male neonatal Sprague-Dawley rats were briefly exposed to 0.1 to 5,000 µg BPA/kg BW on postnatal days (PND) 1, 3, and 5. Individual prostate lobes plus periurethral prostatic ducts were evaluated at 7 mo or 1 y of age without or with adult testosterone plus estradiol (T+E) to promote carcinogenesis. DNA methylation of five genes was quantified by bisulfite genomic sequencing in d-200 dorsal prostates across BPA doses. Serum free-BPA and BPA-glucuronide were quantitated in sera of individual PND 3 pups collected 1 hr postexposure utilizing ultra-high-pressure tandem mass spectrometry (UHPLC-MS-MS). RESULTS: The lowest BPA dose initiated maximal hormonal carcinogenesis in lateral prostates despite undetectable free BPA 1 hr postexposure. Further, prostatic intraepithelial neoplasia (PIN) progressed to carcinoma in rats given neonatal low-dose BPA with adult T+E but not in rats given adult T+E alone. The dorsal and ventral lobes and periurethral prostatic ducts exhibited a nonmonotonic dose response with peak PIN, proliferation and apoptotic values at 10­100 µg/kg BW. This was paralleled by nonmonotonic and dose-specific DNA hypomethylation of genes that confer carcinogenic risk, with greatest hypomethylation at the lowest BPA doses. CONCLUSIONS: Developmental BPA exposures heighten prostate cancer susceptibility in a complex dose- and lobe-specific manner. Importantly, elevated carcinogenic risk is found at doses that yield undetectable serum free BPA. Dose-specific epigenetic modifications of selected genes provide a mechanistic framework that may connect early-life BPA to later-life predisposition to prostate carcinogenesis. https://doi.org/10.1289/EHP1050.

DNA methylome changes by estradiol benzoate and bisphenol A links early-life environmental exposures to prostate cancer risk.

Developmental exposure to endocrine-disrupting chemicals (EDCs), 17ß-estradiol-3-benzoate (EB) and bisphenol A (BPA), increases susceptibility to prostate cancer (PCa) in rodent models. Here, we used the methylated-CpG island recovery assay (MIRA)-assisted genomic tiling and CpG island arrays to identify treatment-associated methylome changes in the postnatal day (PND)90 dorsal prostate tissues of Sprague-Dawley rats neonatally (PND1, 3, and 5) treated with 25 µg/pup or 2,500 µg EB/kg body weight (BW) or 0.1 µg BPA/pup or 10 µg BPA/kg BW. We identified 111 EB-associated and 86 BPA-associated genes, with 20 in common, that have significant differentially methylated regions. Pathway analysis revealed cancer as the top common disease pathway. Bisulfite sequencing validated the differential methylation patterns observed by array analysis in 15 identified candidate genes. The methylation status of 7 (Pitx3, Wnt10b, Paqr4, Sox2, Chst14, Tpd52, Creb3l4) of these 15 genes exhibited an inverse correlation with gene expression in tissue samples. Cell-based assays, using 5-aza-cytidine-treated normal (NbE-1) and cancerous (AIT) rat prostate cells, added evidence of DNA methylation-mediated gene expression of 6 genes (exception: Paqr4). Functional connectivity of these genes was linked to embryonic stem cell pluripotency. Furthermore, clustering analyses using the dataset from The Cancer Genome Atlas revealed that expression of this set of 7 genes was associated with recurrence-free survival of PCa patients. In conclusion, our study reveals that gene-specific promoter methylation changes, resulting from early-life EDC exposure in the rat, may serve as predictive epigenetic biomarkers of PCa recurrence, and raises the possibility that such exposure may impact human disease.

Serum bisphenol A pharmacokinetics and prostate neoplastic responses following oral and subcutaneous exposures in neonatal Sprague-Dawley rats.

The present study examines BPA pharmacokinetics in neonatal rats following s.c. injection or oral delivery of 10 µg BPA/kg BW and compares susceptibility to estrogen-induced prostate intraepithelial neoplasia (PIN) following either exposure route. Serum BPA in PND3 rats was measured using HPLC-MS-MS. Free and total BPA at C(max) were 1.77 and 2.0 ng/ml, respectively following injection and 0.26 and 1.02 ng/ml, respectively following oral exposure. The AUC(0-2) for free and total BPA was 4.1-fold and 1.8-fold greater, respectively, in s.c. vs. oral delivery. While exposure route affected BPA metabolism, internal dosimetry following s.c. injection of 10 µg BPA/kg BW is similar to BPA levels observed in humans. Prostates from aged rats given s.c. or oral BPA neonatally and T+E implants as adults exhibited nearly identical, heightened susceptibility to PIN incidence and score as compared to neonatal oil-controls. These findings on prostate health are directly relevant to humans at current BPA exposure levels.

Estrogen replacement therapy in diabetic ovariectomized female rats potentiates postischemic leukocyte adhesion in cerebral venules via a RAGE-related process.

In this study, we tested the hypothesis that the documented transformation of 17beta-estradiol (E2) from a counterinflammatory hormone in nondiabetic (ND) rats to a proinflammatory agent in rats with diabetes mellitus (DM) is due to an enhanced contribution from the receptor for advanced glycation end products (RAGE). Rhodamine 6G-labeled leukocytes were observed through a closed cranial window in rats. In vivo pial venular leukocyte adherence and infiltration were measured over 10 h reperfusion after transient forebrain ischemia in DM (streptozotocin) versus ND intact, ovariectomized (OVX), and E2-replaced (for 7-10 days) OVX (OVE) females. The role of RAGE was examined in two ways: 1) RAGE knockdown via topical application of RAGE antisense versus missense oligodeoxynucleotide or 2) intracerebroventricular injection of the RAGE decoy inhibitor, soluble RAGE. Among diabetic rats, the lowest levels of cortical RAGE mRNA and immunoreactivity of the RAGE ligand, AGE, were seen in OVX females, with significantly higher levels exhibited in intact and OVE females. However, results from the analysis of cortical RAGE protein only partially tracked those findings. When comparing ND to DM rats, cortical AGE immunoreactivity was significantly lower in OVE and intact females but similar in OVX rats. In DM rats, the level of postischemic leukocyte adhesion and infiltration (highest to lowest) was OVE>intact>>untreated OVX. In NDs, adhesion was highest in the untreated OVX group. Leukocyte extravasation was observed at >6 h postischemia but only in diabetic OVE and intact females and in ND OVX (untreated) rats. Pretreatment with RAGE antisense-oligodeoxynucleotide or soluble RAGE attenuated postischemic leukocyte adhesion and prevented infiltration but only in the diabetic OVE and intact groups. These results indicate that the exacerbation of postischemic leukocyte adhesion by chronic E2 replacement therapy in diabetic OVX females involves a RAGE-related mechanism. Targeting RAGE may restore the neuroprotective effect of E2 replacement therapy in diabetic females.

Astrocytes are a key conduit for upstream signaling of vasodilation during cerebral cortical neuronal activation in vivo.

Astrocytes play an important role in the coupling between neuronal activity and brain blood flow via their capacity to "sense" neuronal activity and transmit that information to parenchymal arterioles. Here we show another role for astrocytes in neurovascular coupling: the ability to act as a signaling conduit for the vitally important process of upstream vasodilation (represented by pial arterioles) during both excessive (seizure) and physiological (sciatic nerve stimulation) increases in cerebral cortical neuronal activity. The predominance of an astrocytic rather than a vascular route was indicated by data showing that pial arteriolar-dilating responses to neuronal activation were completely blocked following selective disruption of the superficial glia limitans, whereas interference with interendothelial signaling was without effect. Results also revealed contributions from connexin 43, implying a role for gap junctions and/or hemichannels in the signaling process and that signaling from the glia limitans to pial arterioles may involve a diffusible mediator.

The role of the glia limitans in ADP-induced pial arteriolar relaxation in intact and ovariectomized female rats.

We examined whether the glia limitans (GL) influences pial arteriolar relaxation elicited in vivo by the purinergic (P(2)Y(1) receptor) agonist ADP in female rats, and whether that influence is altered in ovariectomized (Ovx) females. A validated model for GL injury was used, topical application of the gliotoxin L-alpha-aminoadipic acid (L-alphaAAA), 24 h before the study. In both intact and Ovx females, L-alphaAAA had no effect on responses to the NO donor, S-nitroso-N-acetyl penicillamine, but ADP-induced pial arteriolar dilations were significantly reduced (by 33-90%), compared with vehicle-treated controls. When N(G)-nitro-L-arginine (L-NNA) was administered to L-alphaAAA-treated rats, the ADP response was virtually lost in intact females, but no further reductions were observed in the Ovx rats. On the other hand, in L-alphaAAA-treated Ovx females, when the gap junction blocker, Gap 27, was subsequently added to the suffusate, ADP reactivity fell to very low levels. In vehicle-treated control rats, L-NNA and Gap 27 reduced ADP reactivity by approximately 50% in intact and Ovx females, respectively. An earlier study indicated that the endothelium was a key site of influence for L-NNA (intact) and Gap 27 (Ovx). Thus present and previous results imply that the ADP response in pial arterioles represents the additive actions of an endothelial and a GL component. That supposition was confirmed in the present study by the finding that combining endothelial and GL injury produced an essentially complete loss of ADP reactivity in both intact and Ovx females. Finally, topical application of the selective P(2)Y(1) antagonist, MRS-2179, was associated with a nearly complete suppression of the ADP response in both intact and Ovx females. These results suggest that 1) ADP-induced pial arteriolar dilation involves additive contributions from P(2)Y(1) receptors present in both vascular endothelium and the GL; 2) the influence of the GL component is not altered by ovariectomy; and 3) the gap junction-dependent component of the ADP response in Ovx females is unlikely to include the GL and probably resides in the vessels themselves.

Loss of benefit from estrogen replacement therapy in diabetic ovariectomized female rats subjected to transient forebrain ischemia.

In nondiabetic animals, estrogen has been shown to provide significant neuroprotection in focal and transient forebrain ischemia models. However, that neuroprotection may be diminished or lost in the diabetic. In this study, we compared the level of brain damage in intact, ovariectomized (OVX) and 17beta-estradiol (E(2))-treated OVX female rats rendered diabetic and chronically ( approximately 4 weeks) hyperglycemic via streptozotocin (STZ). Rats were subjected to 20 min of unilateral transient forebrain ischemia (reduction in cortical CBF to 20% of baseline). Neurologic function was analyzed daily and brain histopathology (in H&E-stained sections) was evaluated at 72 h of reperfusion. Supplemental histopathologic information was obtained from additional TUNEL-stained sections. When comparing neurologic outcome scores in the three groups, E(2)-treated OVX females displayed the highest degree of dysfunction and intact females the least (OVX rats not treated with E(2) were intermediate), with the difference between the intact and E(2)-treated groups being statistically significant. That same order was often observed with the regional histopathologic analyses of H&E-stained tissue. A significantly higher magnitude of neuronal loss in both OVX groups, when compared to intact females, was observed in the CA4 sector of the hippocampus and in the cortex. In addition, cell loss in the dorsal thalamus of the E(2)-treated group was significantly greater than in the intact females. Those results were generally corroborated by TUNEL-analysis, with 67% of the E(2)-treated, 33% of the control OVX, and only 17% of the intact females displaying TUNEL-positive cells in multiple regions. In conclusion, the present findings strongly suggest that the neuroprotective benefits of estrogen replacement therapy may be lost in the diabetic female rat.