Association between asthma and periodontitis: A case-control analysis of risk factors, related medications, and allergic responses.

AIMS: This study aimed to investigate the association between asthma, related allergies and medication use, and the presence and severity of periodontitis among individuals at the University of Michigan School of Dentistry. METHODS: Employing a case-control design, the study analyzed data from 892 patients, half with asthma and half without asthma. Data collection included demographics, asthma history, medication use, allergies, and periodontal examination outcomes, including probing pocket depth (PPD), mobility, furcation involvement, and radiographic bone loss (RBL). Logistic regression models assessed the relationship between asthma and periodontitis, adjusting for confounders. RESULTS: Asthmatic patients exhibited significantly lower odds of periodontitis (OR = 0.10, p < .001) and were less likely to present with advanced stages (OR = 0.23, p < .001) and grades of the disease (OR = 0.31, p < .001) compared to non-asthmatic patients. The study also found a higher proportion of females in the asthmatic group (67% vs. 51.8%, p < .001). Smoking was identified as a significant factor associated with periodontitis in patients with asthma, with former smokers at more than double the odds (OR = 2.28, p = .035) and current smokers at a slightly lower yet significant odds (OR = 1.87, p = .050). Additionally, asthmatic patients on adrenergic inhalers had an increased likelihood of developing periodontitis (OR = 1.76, p = .045). Allergies to codeine and latex were associated with higher odds of periodontitis, with ORs of 3.41 and 6.09, respectively. CONCLUSIONS: Asthma was found to be associated with lower odds of periodontitis. However, this association appears to be modified by smoking habits and the use of certain asthma medications, which are related to an increased likelihood of periodontitis among asthmatic patients.

Periodontitis stage and grade modifies the benefit of regular supportive periodontal care in terms of need for retreatment and mean cumulative cost.

AIM: This study aimed to characterize the periodontal breakdown during supportive periodontal care (SPC) and to quantify the corresponding cost-effectiveness of periodontal therapy. MATERIALS AND METHODS: Data were obtained from charts of patients who received active periodontal therapy (APT) with a minimum follow-up of ≥10 years. Analysis was done to identify factors associated with the incidence of additional sub-gingival instrumentation (SGI) and/or surgery (SUR) during SPC and mean cumulative cost of recurrence was calculated. All relevant data were collected. RESULTS: In all, 442 patients were included. Over the follow-up period, 62% of Stage I and II patients and 72% of Stage III and IV patients required further treatment following the APT; 56.5% of SGI patients and 78.6% of SUR patients received a second intervention. SUR patients received more SUR during the follow-up period (p = .035). Stage III and IV patients received more SUR during SPC than Stage I and II patients (p = .001). Grade C patients received more SUR during the follow-up period (p < .05). During the 5-year period preceding retreatment, the mean SPC visits were lower for patients who did not require retreatment (p < .001). Risk factors such as regularity of maintenance, smoking and diabetes were related to a higher chance of receiving SUR during the follow-up period (p < .05). The mean cumulative costs indicated less recurrence cost for compliers in Stage III and IV or Grade B and C but not for those in Stage I and II or Grade A. CONCLUSIONS: The risk of relapse in the maintenance population may be correlated with higher stage and grade, patient compliance, modifiable risk factors and the nature of the treatment provided during APT. The total cost of treatment of recurrences was lower for compliers in Stage III/IV and Grade B/C compared with erratic compliers with the same severity and risk.

Hyperbaric oxygen therapy as an adjunct treatment of periodontitis, MRONJ, and ONJ: a systematic literature review.

OBJECTIVE: To review the available prospective literature on hyperbaric oxygen (HBO) therapy for periodontal conditions. MATERIALS AND METHODS: A comprehensive electronic and manual search was performed to identify clinical studies on adult patients who underwent hyperbaric oxygen therapy for periodontal treatments. A systematic literature search was conducted in PubMed, Cochrane, and Dentistry Oral Sciences Source databases. RESULTS: Fourteen articles were included in the final literature review, of which five were RCTs and 11 were prospective clinical studies. Four studies discussed HBO as an adjunct to nonsurgical treatment of periodontitis, eight reported on HBO and osteoradionecrosis, and one examined HBO in bisphosphonate-related necrosis of the jaws. CONCLUSIONS: HBO has shown superior efficacy compared to antibiotics as a prophylactic measure in preventing osteoradionecrosis (ORN) in patients with a history of high mandibular irradiation. Clinicians should consider referring such patients for HBO therapy before and after tooth extractions. However, for the surgical excision of existing ORN lesions, HBO therapy does not yield significant benefits but does not negatively impact outcomes either. Regarding the treatment of periodontitis patients, the variability among studies prevents definitive conclusions. HBO therapy as an adjunct to SRP in periodontitis treatment produces mixed results. CLINICAL RELEVANCE: This study's clinical relevance lies in its exploration of the potential benefits of HBO for periodontal conditions. Also, it provides clinicians with insights into when and how to integrate HBO therapy into their treatment approaches, particularly for patients with a history of irradiation and those undergoing complex dental procedures.

Discovery of 1,3-disubstituted pyrazole peripheral cannabinoid receptor partial agonists.

Partial agonists of peripheral cannabinoid receptors (CBRs) have potential therapeutic applications in several medical conditions. However, (-)-trans-Δ9-tetrahydrocannabinol (THC), the principal active component of marijuana, which is a partial agonist of CB1 and CB2 penetrates the central nervous system (CNS) and produces adverse effects. Peripherally restricted partial agonists of CBRs, particularly of CB1, can be used to treat illnesses safely and effectively with a better therapeutic index. Here, we report on our efforts to synthesize pyrazole partial CBR agonists with peripheral selectivity, resulting in lead compound 40. This compound is a potent partial agonist of CB1 with âˆ¼ 5-fold higher plasma biodistribution over brain and represents an early lead for optimization.

Synthesis and pharmacological validation of a novel radioligand for the orphan GPR88 receptor.

GPR88 is an orphan G protein-coupled receptor which has been implicated in a number of striatal-associated disorders. Herein we describe the synthesis and pharmacological characterization of the first GPR88 radioligand, [3H]RTI-33, derived from a synthetic agonist RTI-13951-33. [3H]RTI-33 has a specific activity of 83.4 Ci/mmol and showed one-site, saturable binding (KD of 85 nM) in membranes prepared from stable PPLS-HA-hGPR88-CHO cells. A competition binding assay was developed to determine binding affinities of several known GPR88 agonists. This radioligand represents a powerful tool for future mechanistic and cell-based ligand-receptor interaction studies of GPR88.

How to avoid intraoperative and postoperative complications in maxillary sinus elevation.

Maxillary sinus floor elevation, via the lateral approach, is one of the most predictable bone augmentation procedures performed in implant dentistry. but both intra- and postoperative complications can occur, and some of them are severe. Our aim is as follows: To review the pertinent literature on the topic, especially assessing the risk factors related to complications. To give clinical recommendations to minimize intra- and postoperative complications with the ultimate scope of improving the standard of clinical care and patient safety.

Fluoroalkoxylated C-3 and C-9 (S)-12-bromostepholidine analogues with D1R antagonist activity.

To illuminate the tolerance of fluoroalkoxylated groups at the C-3 and C-9 positions of tetrahydroprotoberberines (THPBs) on D1R activity, C-3 and C-9 fluoroalkoxylated analogues of (S)-12-bromostepholidine were prepared and evaluated. All compounds examined were D1R antagonists as measured by a cAMP assay. Our structure-activity studies herein indicate that the C-3 position tolerates a 1,1-difluoroethoxy substituent for D1R antagonist activity. Compound 13a was the most potent cAMP-based D1R antagonist identified and was also found to antagonize ß-arrestin translocation in a TANGO assay. Affinity assessments at other dopamine receptors revealed that 13a is selective for D1R and unlike other naturally-occurring THPBs such as (S)-stepholidine, lacks D2R affinity. In preliminary biopharmaceutical assays, excellent BBB permeation was observed for 13a. Further pharmacological studies are warranted on (S)-stepholidine congeners to harvest their potential as a source of novel, druggable D1R-targeted agents.

Supplement Consumption and Periodontal Health: An Exploratory Survey Using the BigMouth Repository.

BACKGROUND: Dietary supplements have been investigated for their impact on the periodontal apparatus (alveolar bone, mucosa, periodontal ligament, and cementum) and their hypothetical protective role against periodontitis. There remains a gap in the field in this area. Thus, the present study aims to examine the correlation between populations who report taking different dietary supplements and their relative periodontal health. METHODS: The BigMouth dental data repository derived from the dental Electronic Health Records (EHRs) of the University of Michigan school of dentistry was used to extract data relating to all patients who fulfilled the eligibility criteria. The prevalence of periodontitis compared to periodontal health as related to supplement consumption was assessed. RESULTS: A total of 118,426 individuals (55,459 males and 62,967 females) with self-reported consumption of the dietary supplements of interest were identified in the University of Michigan database via the BigMouth repository. Associations with the following vitamins were investigated, Vitamin B, Vitamin C, Vitamin D, Vitamin E, Multivitamins, Fish oil, Calcium, Omega 3, Saw palmetto, Zinc, Sildenafil, Flax seed, Folic acid, Garlic pills, Ginger pills, Ginko, Ginseng, Glucosamine, Iron, and Magnesium. Out of these supplements, only multivitamins and iron were found to significantly favor periodontal health, while folic acid and vitamin E significantly favored periodontitis. CONCLUSIONS: This study found a minimal association between the consumption of dietary supplements with periodontal health.

The GPR88 agonist RTI-13951-33 reduces alcohol drinking and seeking in mice.

GPR88 is an orphan G-protein-coupled receptor that is considered a potential target to treat neuropsychiatric disorders, including addiction. Most knowledge about GPR88 function stems from knockout mouse studies, and in vivo pharmacology is still scarce. Here we examine the effects of the novel brain-penetrant agonist RTI-13951-33 on several alcohol-related behaviours in the mouse. In the intermittent-access-two-bottle-choice paradigm, the compound reduced excessive voluntary alcohol drinking, while water drinking was intact. This was observed for C57BL/6 mice, as well as for control but not Gpr88 knockout mice, demonstrating efficacy and specificity of the drug in vivo. In the drinking-in-the-dark paradigm, RTI-13951-33 also reduced binge-like drinking behaviour for control but not Gpr88 knockout mice, confirming the alcohol consumption-reducing effect and in vivo specificity of the drug. When C57BL/6 mice were trained for alcohol self-administration, RTI-13951-33 decreased the number of nose-pokes over a 4-h session and reduced the number of licks and bursts of licks, suggesting reduced motivation to obtain alcohol. Finally, RTI-13951-33 did not induce any place preference or aversion but reduced the expression of conditioned place preference to alcohol, indicative of a reduction of alcohol-reward seeking. Altogether, data show that RTI-13951-33 limits alcohol intake under distinct conditions that require consummatory behaviour, operant response or association with contextual cues. RTI-13951-33 therefore is a promising lead compound to evaluate GPR88 as a therapeutic target for alcohol use disorders. More broadly, RTI-13951-33 represents a unique tool to better understand GPR88 function, disentangle receptor roles in development from those in the adult and perhaps address other neuropsychiatric disorders.

Trueness and Precision of Economical Smartphone-Based Virtual Facebow Records.

PURPOSE: To investigate the trueness and precision of virtual facebow records using a smartphone as a three-dimensional (3D) face scanner. MATERIAL AND METHODS: Twenty repeated virtual facebow records were performed on two subjects using a smartphone as a 3D face scanner. For each subject, a virtual facebow was attached to his/her maxillary arch, and face scans were performed using a smartphone with a 3D scan application. The subject's maxillary arch intraoral scan was aligned to the face scan by the virtual facebow fork. This procedure was repeated 10 times for each subject. To investigate if the maxillary scan is located at the right position to the face, these virtual facebow records were superimposed to a cone-beam computed tomography (CBCT) head scan from the same subject by matching the face scan to the 3D face reconstruction from CBCT images. The location of maxillary arch in virtual facebow records was compared with its position in CBCT. The "trueness" of the proposed procedure is defined as the deviation between maxilla arch position in virtual facebow records and the CBCT images. The "precision" is defined as the deviation between each virtual facebow record. The linear deviation at left central incisor (#9), left first molar (#14), and right first molar (#3), as well as angular deviation of occlusal plane were analyzed with descriptive statistics. Differences between two objects were also explored with Mann Whitney U test. RESULTS: The 20 virtual facebow records using the smartphone 3D scanner deviated from the CBCT measurements (trueness) by 1.14 ± 0.40 mm at #9, 1.20 ± 0.50 mm at #14, 1.12 ± 0.51 mm at the #3, and 1.48 ± 0.56° in the occlusal plane. The VFTs deviated from each other by 1.06 ± 0.50 mm at #9, 1.09 ± 0.49 mm at #14, 1.11 ± 0.58 mm at #3, and 0.81 ± 0.58° in the occlusal plane. When all sites combined, the trueness was 1.14 ± 0.40 mm, and the precision was 1.08 ± 0.52 mm. Out of eight measurements, three measurements were significantly different between subjects. Nevertheless, the mean difference was small. CONCLUSIONS: Virtual facebow records made using smartphone-based face scan can capture the maxilla position with high trueness and precision. The deviation can be anticipated as around 1 mm in linear distance and 1° in angulation.

The psychobiological links between chronic stress-related diseases, periodontal/peri-implant diseases, and wound healing.

Chronic stress is a relevant disease to periodontal practice, encompassing 25%-28% of the US population (American Psychological Association 2015). While it is well established that chronic psychologic stress can have significant deleterious systemic effects, only in recent decades have we begun to explore the biochemical, microbial, and physiologic impacts of chronic stress diseases on oral tissues. Currently, chronic stress is classified as a "risk indicator" for periodontal disease. However, as the evidence in this field matures with additional clinically controlled trials, more homogeneous data collection methods, and a better grasp of the biologic underpinnings of stress-mediated dysbiosis, emerging evidence suggests that chronic stress and related diseases (depression, anxiety) may be significant contributing factors in periodontal/peri-implant disease progression and inconsistent wound healing following periodontal-related therapeutics. Ideal solutions for these patients include classification of the disease process and de-escalation of chronic stress conditions through coping strategies. This paper also summarizes periodontal/implant-related therapeutic approaches to ensure predictable results for this specific patient subpopulation.

Rational design of cannabinoid type-1 receptor allosteric modulators: Org27569 and PSNCBAM-1 hybrids.

Allosteric modulation offers an alternate approach to target the cannabinoid type-1 receptor (CB1) for therapeutic benefits. Examination of the two widely studied prototypic CB1 negative allosteric modulators (NAMs) Org27569 and PSNCBAM-1 revealed structural resemblance and similar structure-activity relationships (SARs). In silico docking and dynamics simulation studies using the crystal structure of CB1 co-bound with CP55,940 and Org27569 suggested that Org27569 and PSNCBAM-1 occupied the same binding pocket and several common interactions were present in both series with the CB1 receptor. A new scaffold was therefore designed by merging the key structural features from the two series and the hybrids retained these binding features in the in silico docking studies. In addition, one such hybrid displayed similar functions to Org27569 in dynamic simulations by preserving a key R2143.50-D3386.30 salt bridge and maintaining an antagonist-like Helix3-Helix6 interhelical distance. Based on these results, a series of hybrids were synthesized and assessed in calcium mobilization, [35S]GTPγS binding and cAMP assays. Several compounds displayed comparable potencies to Org27569 and PSNCBAM-1 in these assays. This work offers new insight of the SAR requirement at the allosteric site of the CB1 receptor and provides a new scaffold that can be optimized for the development of future CB1 allosteric modulators.

CXCL12γ induces human prostate and mammary gland development.

BACKGROUND: Epithelial stem cells (ESCs) demonstrate a capacity to maintain normal tissues homeostasis and ESCs with a deregulated behavior can contribute to cancer development. The ability to reprogram normal tissue epithelial cells into prostate or mammary stem-like cells holds great promise to help understand cell of origin and lineage plasticity in prostate and breast cancers in addition to understanding normal gland development. We previously showed that an intracellular chemokine, CXCL12γ induced cancer stem cells and neuroendocrine characteristics in both prostate and breast adenocarcinoma cell lines. However, its role in normal prostate or mammary epithelial cell fate and development remains unknown. Therefore, we sought to elucidate the functional role of CXCL12γ in the regulation of ESCs and tissue development. METHODS: Prostate epithelial cells (PNT2) or mammary epithelial cells (MCF10A) with overexpressed CXCL12γ was characterized by quantitative real-time polymerase chain reaction, Western blots, and immunofluorescence for lineage marker expression, and fluorescence activated cell sorting analyses and sphere formation assays to examine stem cell surface phenotype and function. Xenotransplantation animal models were used to evaluate gland or acini formation in vivo. RESULTS: Overexpression of CXCL12γ promotes the reprogramming of cells with a differentiated luminal phenotype to a nonluminal phenotype in both prostate (PNT2) and mammary (MCF10A) epithelial cells. The CXCL12γ-mediated nonluminal type cells results in an increase of epithelial stem-like phenotype including the subpopulation of EPCAMLo /CD49fHi /CD24Lo /CD44Hi cells capable of sphere formation. Critically, overexpression of CXCL12γ promotes the generation of robust gland-like structures from both prostate and mammary epithelial cells in in vivo xenograft animal models. CONCLUSIONS: CXCL12γ supports the reprogramming of epithelial cells into nonluminal cell-derived stem cells, which facilitates gland development.

Bone microenvironment signaling of cancer stem cells as a therapeutic target in metastatic prostate cancer.

Prostate cancer (PCa) is one of the most prevalent cancers and the second leading cause of cancer death among US males. When diagnosed in an early disease stage, primary tumors of PCa may be treated with surgical resection or radiation, sometimes combined with androgen deprivation therapy, with favorable outcomes. Unfortunately, the treatment efficacy of each approach decreases significantly in later stages of PCa that involve metastasis to soft tissues and bone. Metastatic PCa is a heterogeneous disease containing host cells, mature cancer cells, and subpopulation of cancer stem cells (CSC). CSCs are highly tumorigenic due to their self-renewing and differentiating potential, clinically resulting in recurrence and resistance to standard therapies. Therefore, there is a large unmet clinical need to develop therapies, which target CSC activity. In this review, we summarize the main signaling pathways that are implicated in the current pre-clinical and clinical studies of recurrent metastatic PCa within the bone microenvironment targeting CSCs and discuss the trajectory of therapeutics moving forward.

The assessment of stress, depression, and inflammation as a collective risk factor for periodontal diseases: a systematic review.

OBJECTIVES: The purpose of this review was to provide a novel perspective utilizing an assessment of biomarkers to evaluate the impact of stress-related disorders on the progression of periodontal disease and evaluate the growing body of evidence of stress as a risk indicator for periodontal disease progression. METHODS: Cross-sectional, case-control, and biomarker studies associating psychological disorders and periodontal disease were included in the literature search. Computational studies, animal studies, reviews, and studies lacking healthy controls were excluded. Electronic and manual literature searches were conducted by two independent reviewers in several databases as well as a manual search for relevant articles published up to January 2018. RESULTS: Twenty-six articles fulfilled the inclusion criteria and were included in the qualitative synthesis. Relationships between stress-related disorders and serum and salivary biomarkers such as cortisol, dehydroepiandrosterone (DHEA), chromogranin A (CgA), and pro-inflammatory cytokines were identified. CONCLUSIONS: The use of salivary pro-inflammatory cytokines alone is not sufficient for the identification of periodontal disease severity/progression with or without the presence of stress-associated diseases. Keeping in mind the limitations of this review, a positive qualitative correlation was observed in the literature among stress-related biomarkers and the severity of periodontal disease. This correlation may serve as an important reporter of patient susceptibility for periodontal breakdown in the future. CLINICAL RELEVANCE: Stress-related disorders should be included in the list of globally screened diseases because it can change the biochemistry of both the local periodontal microenvironment as well as the global systemic inflammatory burden.

Detection and isolation of disseminated tumor cells in bone marrow of patients with clinically localized prostate cancer.

BACKGROUND: Disseminated tumor cells (DTCs) have been reported in the bone marrow (BM) of patients with localized prostate cancer (PCa). However, the existence of these cells continues to be questioned, and few methods exist for viable DTC isolation. Therefore, we sought to develop novel approaches to identify and, if detected, analyze localized PCa patient DTCs. METHODS: We used fluorescence-activated cell sorting (FACS) to isolate a putative DTC population, which was negative for CD45, CD235a, alkaline phosphatase, and CD34, and strongly expressed EPCAM. We examined tumor cell content by bulk cell RNA sequencing (RNA-Seq) and whole-exome sequencing after whole genome amplification. We also enriched for BM DTCs with α-EPCAM immunomagnetic beads and performed quantitative reverse trancriptase polymerase chain reaction (qRT-PCR) for PCa markers. RESULTS: At a threshold of 4 cells per million BM cells, the putative DTC population was present in 10 of 58 patients (17%) with localized PCa, 4 of 8 patients with metastatic PCa of varying disease control, and 1 of 8 patients with no known cancer, and was positively correlated with patients' plasma PSA values. RNA-Seq analysis of the putative DTC population collected from samples above (3 patients) and below (5 patients) the threshold of 4 putative DTCs per million showed increased expression of PCa marker genes in 4 of 8 patients with localized PCa, but not the one normal donor who had the putative DTC population present. Whole-exome sequencing also showed the presence of single nucleotide polymorphisms and structural variants in the gene characteristics of PCa in 2 of 3 localized PCa patients. To examine the likely contaminating cell types, we used a myeloid colony formation assay, differential counts of cell smears, and analysis of the RNA-Seq data using the CIBERSORT algorithm, which most strongly suggested the presence of B-cell lineages as a contaminant. Finally, we used EPCAM enrichment and qRT-PCR for PCa markers to estimate DTC prevalence and found evidence of DTCs in 21 of 44 samples (47%). CONCLUSION: These data support the presence of DTCs in the BM of a subset of patients with localized PCa and describe a novel FACS method for isolation and analysis of viable DTCs.

The Effect of Implant-Induced Artifacts on Interpreting Adjacent Bone Structures on Cone-Beam Computed Tomography Scans.

INTRODUCTION: Cone-beam computed tomography (CBCT) imaging can be used to visualize anatomical structures before implant placement. The aim of this study is to evaluate the impact of implant artifacts on the accuracy of measuring periimplant bone dimensions. MATERIALS AND METHODS: Nineteen implants were placed into 9 fresh, frozen cadavers. A CBCT scan was taken, the implants were removed, and a second scan was taken. Implant dimensions and periimplant bone measurements were calculated. The mean differences were compared with paired t tests. Pearson's correlation coefficients were calculated for bone thickness measurements. DISCUSSION: No significant differences were found between the implant dimension or bone thickness measurements on each scan. Bone thickness at the implant platform and apex were significantly correlated (P < 0.001). CONCLUSION: The presence of dental implants did not impact the accuracy of cone-beam computed tomography (CBCT) measurements of bone thickness by metallic artifacts.

Mer Tyrosine Kinase Regulates Disseminated Prostate Cancer Cellular Dormancy.

Many prostate cancer (PCa) recurrences are thought to be due to reactivation of disseminated tumor cells (DTCs). We previously found a role of the TAM family of receptor tyrosine kinases TYRO3, AXL, and MERTK in PCa dormancy regulation. However, the mechanism and contributions of the individual TAM receptors is largely unknown. Knockdown of MERTK, but not AXL or TYRO3 by shRNA in PCa cells induced a decreased ratio of P-Erk1/2 to P-p38, increased expression of p27, NR2F1, SOX2, and NANOG, induced higher levels of histone H3K9me3 and H3K27me3, and induced a G1/G0 arrest, all of which are associated with dormancy. Similar effects were also observed with siRNA. Most importantly, knockdown of MERTK in PCa cells increased metastasis free survival in an intra-cardiac injection mouse xenograft model. MERTK knockdown also failed to inhibit PCa growth in vitro and subcutaneous growth in vivo, which suggests that MERTK has specificity for dormancy regulation or requires a signal from the PCa microenvironment. The effects of MERTK on the cell cycle and histone methylation were reversed by p38 inhibitor SB203580, which indicates the importance of MAP kinases for MERTK dormancy regulation. Overall, this study shows that MERTK stimulates PCa dormancy escape through a MAP kinase dependent mechanism, also involving p27, pluripotency transcription factors, and histone methylation. J. Cell. Biochem. 118: 891-902, 2017. © 2016 Wiley Periodicals, Inc.

Human Stem Cells Overexpressing miR-21 Promote Angiogenesis in Critical Limb Ischemia by Targeting CHIP to Enhance HIF-1α Activity.

Critical limb ischemia (CLI) is a severe blockage in the arteries of the lower extremities. However, the effective and optimal treatment for CLI remains to be elucidated. Previous therapeutic research is mainly focused on proangiogenic growth factors administrations. Recently, miR-21 has been revealed to play a crucial role in angiogenesis. Thus, we hypothesize that miR-21 over-expression in human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) can effectively treat CLI. Herein, UCBMSCs were transduced with lentivirus-miR-21-Luciferase (Lenti-miR-21) or lentivirus- LacZ-Luciferase (Lenti-LacZ). The results indicated that miR-21 induced UCBMSCs proliferation, migration, and angiogenesis in vitro. Subsequently, general observation and laser Doppler perfusion imaging were introduced to detect perfusion in muscles of CLI-nude mice on 1, 4, 7, 14, and 28 day postoperation. There was a significant improvement in blood vessels of the ischemic limb in Lenti-miR-21 group at 7 day compared with the saline or Lenti-LacZ groups. At 28 day, histological analysis confirmed that UCBMSCs over-expressing miR-21 increased neovascularization in CLI. Furthermore, carboxyl terminus of Hsc70-interacting protein (CHIP) was found to be the target gene for miR-21-mediated activation of hypoxia-inducible factor 1α (HIF-1α) in UCBMSCs. In summary, our study demonstrated that over-expressing miR-21 in UCBMSCs could improve neovascularization in CLI through enhancing HIF-1α activity by targeting CHIP, which may hold great therapeutic promise in treating CLI.

Development and validation of a high-throughput calcium mobilization assay for the orphan receptor GPR88.

BACKGROUND: GPR88 is an orphan G protein-coupled receptor highly expressed in the striatum and is implicated in basal ganglia-associated disorders. However, the receptor functions of GPR88 are still largely unknown due to the lack of potent and selective ligands appropriate for central nervous system investigation. Development of a high-throughput screening assay for GPR88 should facilitate the discovery of novel ligands to probe GPR88 functions. METHODS: In this paper, we describe the development of a CHO-Gαqi5-GPR88 cell-based calcium mobilization assay. The assay takes advantage of functional coupling of GPR88 with the promiscuous Gαqi5 protein and consequent mobilization of intracellular calcium, which can be measured in a 384-well format with a Fluorescent Imaging Plate Reader. RESULTS: The CHO-Gαqi5-GPR88 cell-based calcium mobilization assay was validated by the structure-activity relationship study of known GPR88 agonist (1R,2R)-2-PCCA analogues. The assay was automated and miniaturized to a 384-well format, and was deemed robust and reproducible with a Z'-factor of 0.72 and tolerated dimethyl sulfoxide to a final concentration of 2%. Screening a pilot neurotransmitter library consisting of 228 compounds yielded 10 hits, but none of the hits were confirmed as GPR88 agonists in follow-up assays. CONCLUSIONS: We have developed a high-throughput calcium mobilization assay for the orphan receptor GPR88. This calcium mobilization assay can be used to identify several different types of GPR88 ligands including agonists, competitive and noncompetitive antagonists, inverse agonists, and allosteric modulators. These ligands will serve as valuable tools to probe signaling mechanisms and in vivo functions of GPR88, and could expedite development of novel therapies for diseases potentially mediated by GPR88.