The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling

The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12(–/–) OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12(–/–), OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent α(IIb)β(3) integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin β(3) gene knockout (Itgb3(–/–)) OCs, but its effects were substantially blunted in P2ry12(–/–) OCs. In vivo, P2ry12(–/–) mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss

Impact of COVID-19 on cardiovascular testing in the United States versus the rest of the world

Objectives: This study sought to quantify and compare the decline in volumes of cardiovascular procedures between the United States and non-US institutions during the early phase of the coronavirus disease-2019 (COVID-19) pandemic. Background: The COVID-19 pandemic has disrupted the care of many non-COVID-19 illnesses. Reductions in diagnostic cardiovascular testing around the world have led to concerns over the implications of reduced testing for cardiovascular disease (CVD) morbidity and mortality. Methods: Data were submitted to the INCAPS-COVID (International Atomic Energy Agency Non-Invasive Cardiology Protocols Study of COVID-19), a multinational registry comprising 909 institutions in 108 countries (including 155 facilities in 40 U.S. states), assessing the impact of the COVID-19 pandemic on volumes of diagnostic cardiovascular procedures. Data were obtained for April 2020 and compared with volumes of baseline procedures from March 2019. We compared laboratory characteristics, practices, and procedure volumes between U.S. and non-U.S. facilities and between U.S. geographic regions and identified factors associated with volume reduction in the United States. Results: Reductions in the volumes of procedures in the United States were similar to those in non-U.S. facilities (68% vs. 63%, respectively; p = 0.237), although U.S. facilities reported greater reductions in invasive coronary angiography (69% vs. 53%, respectively; p < 0.001). Significantly more U.S. facilities reported increased use of telehealth and patient screening measures than non-U.S. facilities, such as temperature checks, symptom screenings, and COVID-19 testing. Reductions in volumes of procedures differed between U.S. regions, with larger declines observed in the Northeast (76%) and Midwest (74%) than in the South (62%) and West (44%). Prevalence of COVID-19, staff redeployments, outpatient centers, and urban centers were associated with greater reductions in volume in U.S. facilities in a multivariable analysis. Conclusions: We observed marked reductions in U.S. cardiovascular testing in the early phase of the pandemic and significant variability between U.S. regions. The association between reductions of volumes and COVID-19 prevalence in the United States highlighted the need for proactive efforts to maintain access to cardiovascular testing in areas most affected by outbreaks of COVID-19 infection

Sustained Radiosensitization of Hypoxic Glioma Cells after Oxygen Pretreatment in an Animal Model of Glioblastoma and <i>In Vitro</i> Models of Tumor Hypoxia

<div><p>Glioblastoma multiforme (GBM) is the most common and lethal form of brain cancer and these tumors are highly resistant to chemo- and radiotherapy. Radioresistance is thought to result from a paucity of molecular oxygen in hypoxic tumor regions, resulting in reduced DNA damage and enhanced cellular defense mechanisms. Efforts to counteract tumor hypoxia during radiotherapy are limited by an attendant increase in the sensitivity of healthy brain tissue to radiation. However, the presence of heightened levels of molecular oxygen during radiotherapy, while conventionally deemed critical for adjuvant oxygen therapy to sensitize hypoxic tumor tissue, might not actually be necessary. We evaluated the concept that pre-treating tumor tissue by transiently elevating tissue oxygenation prior to radiation exposure could increase the efficacy of radiotherapy, even when radiotherapy is administered after the return of tumor tissue oxygen to hypoxic baseline levels. Using nude mice bearing intracranial U87-luciferase xenografts, and <i>in vitro</i> models of tumor hypoxia, the efficacy of oxygen pretreatment for producing radiosensitization was tested. Oxygen-induced radiosensitization of tumor tissue was observed in GBM xenografts, as seen by suppression of tumor growth and increased survival. Additionally, rodent and human glioma cells, and human glioma stem cells, exhibited prolonged enhanced vulnerability to radiation after oxygen pretreatment <i>in vitro</i>, even when radiation was delivered under hypoxic conditions. Over-expression of HIF-1α reduced this radiosensitization, indicating that this effect is mediated, in part, via a change in HIF-1-dependent mechanisms. Importantly, an identical duration of transient hyperoxic exposure does not sensitize normal human astrocytes to radiation <i>in vitro</i>. Taken together, these results indicate that briefly pre-treating tumors with elevated levels of oxygen prior to radiotherapy may represent a means for selectively targeting radiation-resistant hypoxic cancer cells, and could serve as a safe and effective adjuvant to radiation therapy for patients with GBM.</p></div

Normoxic pre-treatment sensitizes glioma cells to radiation after graded chronic hypoxic (GCH) exposure.

<p>(A) The graded chronic hypoxia (GCH) protocol is shown, depicting the timing and severity of hypoxic exposure to four cell lines. Cells either remain in a continuous hypoxic environment (–) or are transiently (25 min) exposed to normoxia 25 min prior to radiation (+). Continuously normoxic cells (NOx) were irradiated as a positive control. (B) The results of anchorage-independent colony forming assays are shown for U87, U87-luc, GL261 glioma cells and 0308 GSCs after 5 Gy radiation exposure under varying oxygen conditions. To allow for ease of comparisons among cell types, raw values are expressed as a percentage of the corresponding cell type’s negative (non-irradiated) control and the means and SEMs are plotted. Each result represents at least three independent samples, plated in triplicate. Holm-Sidak comparisons for multiple groups were used for statistical comparisons of raw values (*p<0.05, **p<0.01). Also shown are Western blots of nuclear HIF-1α at the time of irradiation for each cell type. Corresponding Western blots of lamin A/C are shown as a loading control.</p

The duration of enhanced radiosensitivity after normoxic pretreatment differs among cell lines undergoing Graded Chronic Hypoxia.

<p>(A) The GCH protocol is shown with variable delays to radiation after normoxic pretreatment. After GCH, all cells are transiently (25 min) exposed to normoxia for 25 min (+) and are then returned to severe hypoxia (1% O₂) for 1, 3, or 6 hours prior to radiation. Continuously normoxic cells (NOx) were irradiated as a positive control. (B) Results from anchorage-independent colony forming assays indicate that the decay of enhanced radiosensitivity differs among cell lines. To allow for ease of comparisons among cell types, raw values are expressed as a percentage of the corresponding cell type’s negative (non-irradiated) control and the means and SEMs are plotted. Each result represents at least three independent samples, plated in triplicate. Holm-Sidak comparisons for multiple groups were used for statistical comparisons of raw values (*p<0.05, **p<0.01). Also shown are Western blots of nuclear HIF-1α at the time of irradiation for each cell line. Corresponding Western blots of lamin A/C are shown as a loading control.</p

Normoxic pretreatment sensitizes glioma cells to radiation after rapid acute hypoxic (RAH) exposure.

<p>(A) The rapid acute hypoxia (RAH) protocol is shown depicting the timing and severity of hypoxic exposure. Cells either remain in a continuous hypoxic environment (–) or are transiently (25 min) exposed to normoxia 25 min prior to radiation (+). Continuously normoxic cells (NOx) were irradiated as a positive control. (B) The results of anchorage-independent colony forming assays are shown for U87, U87-luc, GL261 glioma cells and 0308 GSCs after 5 Gy radiation exposure under varying oxygen conditions. To allow for ease of comparisons among cell types, raw values are expressed as a percentage of the corresponding cell type’s negative (non-irradiated) control and the means and SEMs are plotted. Each result represents at least three independent samples, plated in triplicate. Holm-Sidak comparisons for multiple groups were used for statistical comparisons of raw values (**p<0.01). Also shown are Western blots of nuclear HIF-1α at the time of irradiation for each cell type. Corresponding Western blots of lamin A/C are shown as a loading control. All lanes shown that are non-adjacent to the negative control (NOx) are denoted with a separating black line.</p

HIF-1α overexpression rescues oxygen-induced radioresistance in RAH-treated cells, but not GCH-treated cells.

<p>Results are shown for the anchorage-independent colony forming assays for U87 cells transfected with either an empty vector or HIF-1α expression vector and then exposed to GCH or RAH protocols without (–) or with (+) reoxygenation. Continuously normoxic cells (NOx) were irradiated as a positive control. To allow for ease of comparisons among conditions, raw values are presented as a percentage of that cell type’s negative (non-irradiated) control and the means and SEMs are plotted. Each result represents at least three independent samples, plated in triplicate. Holm-Sidak comparisons for multiple groups were used for statistical comparisons of raw values (*p<0.05, **p<0.01). Western blotting analysis of nuclear HIF-1α at the time of irradiation is shown for each cell type below clonogenic results. Corresponding Western blots of lamin A/C are shown as a loading control and blots for hemagglutinin (HA) are shown below HIF-1α overexpression vector results to demonstrate transfection efficacy. All lanes shown that are non-adjacent to the negative control (NOx) are denoted with a separating black line.</p

100% FiO₂ elevates tpO₂ in tumor and healthy brain tissue.

<p>(A) Contrast-enhanced MRI of a mouse brain that was implanted 14 days prior with U87-luc glioma cells is shown. The drawings on the MRI depict the placements of Licox probes used for measuring oxygen levels in the tumor and contralateral brain (striatum). (B and C) The time courses of partial tissue oxygen levels (tpO₂) in response to modified FiO₂ are shown for tumor (triangles) and contralateral (circles) tissue (n = 3). Oxygen measurements are displayed as a percentage of the baseline tpO₂ levels recorded in the contralateral brain. In B, the time courses are plotted using a common ordinate, in order to show the relative levels and changes in tpO₂ levels observed in tumor and contralateral brain. In C, the time courses for tumor and contralateral brain are shown on individual ordinates in order to highlight the difference in the rate of change of oxygen levels between the two tissues. Values shown are means and SEMs. Although radiation was not administered in this experiment, the arrow denoting time of radiation delivery provides a reference for subsequent experiments.</p

Hyperoxic pretreatment improves survival and slows tumor growth.

<p>(A) Surviving fractions of nude mice implanted with U87-luc cells are shown in a Kaplan-Meier plot for animals receiving: no treatment (0 Gy+21%O₂, n = 13), hyperoxic pretreatment without radiation (0 Gy+100%O₂, n = 10), radiation alone (8 Gy+21%O₂, n = 19), and hyperoxic pretreatment with radiation (8 Gy+100%O₂, n = 22). Statistical significance was calculated using a Log-Rank (Mantel-Cox) test. (B) A bar graph shows tumor growth rates for animals in the 8 Gy+21%O₂ group and the 8 Gy+100%O₂ group. Tumor size was assessed by IVIS imaging <i>in vivo</i> performed every 4–5 days. The log of the radiance from up to eight IVIS time points (day 14 to day 46 PTI, depending on individual survival) for each animal was used to perform linear regression analyses of tumor growth. Calculated slopes from these linear regressions were then used to determine average tumor growth rate per day for each animal from each treatment group. Statistical significance was calculated using t-test with Welch’s correction for unequal variance. Error bars are SEMs. (C) IVIS images taken at day 14 and day 31 PTI for two animals, one from the 8 Gy+21%O₂ (left) and one from the 8 Gy+100%O₂ (right) groups. The scale bar for IVIS radiance is shown between the images for the two animals. Note that the IVIS signal in these images does not directly correspond to tumor size. A low luminescence signal threshold was used to permit comparison between time points.</p