Cachexia, characterized by muscle wasting, is a major contributor to cancer-related mortality. However, the key cachexins that mediate cancer-induced muscle wasting remain elusive. Here, we show that tumor-released extracellular Hsp70 and Hsp90 are responsible for tumor’s capacity to induce muscle wasting. We detected high-level constitutive release of Hsp70 and Hsp90 associated with extracellular vesicles (EVs) from diverse cachexia-inducing tumor cells, resulting in elevated serum levels in mice. Neutralizing extracellular Hsp70/90 or silencing Hsp70/90 expression in tumor cells abrogates tumor-induced muscle catabolism and wasting in cultured myotubes and in mice. Conversely, administration of recombinant Hsp70 and Hsp90 recapitulates the catabolic effects of tumor. In addition, tumor-released Hsp70/90-expressing EVs are necessary and sufficient for tumor-induced muscle wasting. Further, Hsp70 and Hsp90 induce muscle catabolism by activating TLR4, and are responsible for elevation of circulating cytokines. These findings identify tumor-released circulating Hsp70 and Hsp90 as key cachexins causing muscle wasting in mice.

Background Microbubbles (MB) combined with ultrasound (US) have been shown to lyse clots without tissue plasminogen activator (tPA) both in vitro and in vivo. We evaluated sonothrombolysis with three types of MB using a rabbit embolic stroke model. Methods New Zealand White rabbits (n=74) received internal carotid angiographic embolization of single 3 day-old cylindrical clots (0.6×4.0-mm). Groups included: 1) control (n=11) embolized without treatment, 2) tPA (n=20), 3) tPA+US (n=10), 4) Perflutren Lipid MB+US (n=16), 5) albumin 3µm MB+US (n=8), and 6) tagged albumin 3µm MB+US (n=9). Treatment began 1 hour post-embolization. Ultrasound was pulsed-wave (1 MHz; 0.8 W/cm2) for 1 hour; rabbits with tPA received intravenous tPA (0.9 mg/kg) over 1 hour. Lipid MB dose was intravenous (0.16 mg/kg) over 30 minutes. Dosage of 3µm MB was 5×109 MB intravenously alone or tagged with eptifibatide and fibrin antibody over 30 minutes. Rabbits were euthanized at 24 hours. Infarct volume was determined using vital stains on brain sections. Hemorrhage was evaluated on H&E sections. Results Infarct volume percent was lower for rabbits treated with Lipid MB+US (1.0%±0.6%; P=0.013), 3µm MB+US (0.7%±0.9%; P=0.018), and tagged 3µm MB+US (0.8%±0.8%; P=0.019) compared with controls (3.5%±0.8%). The three MB types collectively had lower infarct volumes (P=0.0043) than controls. Infarct volume averaged 2.2%±0.6% and 1.7%±0.8% for rabbits treated with tPA alone and tPA+US, respectively (P=NS). Conclusions Sonothrombolysis without tPA using these MB is effective in decreasing infarct volumes. Study of human application and further MB technique development are justified.

Introduction Tissue plasminogen activator (tPA) is the thrombolytic standard of care for acute ischemic stroke, but intracerebral hemorrhage (ICH) remains a common and devastating complication. We investigated using ultrasound (US) and microbubble (MB) techniques to reduce required tPA doses and to decrease ICH. Materials and Methods Fresh blood clots (3–5 hours) were exposed in vitro to tPA (0.02 or 0.1 mg/mL) plus pulsed 1 MHz US (0.1 W/cm2), with or without 1.12 × 108/mL MBs (Definity or albumin/dextrose MBs [adMB]). Clot mass loss was measured to quantify thrombolysis. New Zealand white rabbits (n = 120) received one 3- to 5-hour clot angiographically delivered into the internal carotid artery. All had transcutaneous pulsed 1 MHz US (0.8 W/cm2) for 60 minutes and intravenous tPA (0.1– 0.9 mg/kg) with or without Definity MBs (0.16 mL/mg/kg). After killing the animals, the brains were removed for histology 24 hours later. Results In vitro, MBs (Definity or adMB) increased US-induced clot loss significantly, with or without tPA (P < 0.0001). At 0 and 0.02 mg/mL, tPA clot loss was greater with adMBs compared with Definity (P ≤ 0.05). With MB, the tPA dose was reduced 5-fold with good efficacy. In vivo, both Definity MB and tPA groups had less infarct volume compared with controls at P < 0.0183 and P < 0.0003, respectively. Definity MB+tPA reduces infarct volume compared with controls (P < 0.0001), and ICH incidence outside of strokes was significantly lower (P = 0.005) compared with no MB. However, infarct volume in Definity MB versus tPA was not different at P = 0.19. Conclusion Combining tPAand MB yielded effective loss of clot with very low dose or even no dose tPA, and infarct volumes and ICH were reduced in acute strokes in rabbits. The ability of MBs to reduce tPA requirements may lead to lower rates of hemorrhage in human stroke treatment.

Recent studies have found that those who suffer from posttraumatic stress disorder (PTSD) are more likely to experience dementia as they age, most often Alzheimer's disease (AD). These findings suggest that the symptoms of PTSD might have an exacerbating effect on AD progression. AD and PTSD might also share common susceptibility factors such that those who experience trauma-induced disease were already more likely to succumb to dementia with age. Here, we explored these two hypotheses using a mouse model of PTSD in wild-type and AD model animals. We found that expression of human familial AD mutations in amyloid precursor protein and presenilin 1 leads to sensitivity to trauma-induced PTSD-like changes in behavioral and endocrine stress responses. PTSD-like induction, in turn, chronically elevates levels of CSF ␤-amyloid (A␤), exacerbating ongoing AD pathogenesis. We show that PTSD-like induction and A␤ elevation are dependent on corticotropin-releasing factor (CRF) receptor 1 signaling and an intact hypothalamic-pituitary-adrenal axis. Furthermore, we show that A␤ species can hyperexcite CRF neurons, providing a mechanism by which A␤ influences stress-related symptoms and PTSD-like phenotypes. Consistent with A␤ causing excitability of the stress circuitry, we attenuate PTSD-like phenotypes in vivo by lowering A␤ levels during PTSD-like trauma exposure. Together, these data demonstrate that exposure to PTSD-like trauma can drive AD pathogenesis, which directly perturbs CRF signaling, thereby enhancing chronic PTSD symptoms while increasing risk for AD-related dementia.

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The relative ability of three obesity indices to predict hypertension (HTN) and diabetes (DM) and the validity of using Asian-specific thresholds of these indices were examined in Filipino-American women (FAW). Filipino-American women (n = 382), 40–65 years of age were screened for hypertension (HTN) and diabetes (DM) in four major US cities. Body mass index (BMI), waist circumference (WC) and waist circumference to height ratio (WHtR) were measured. ROC analyses determined that the three obesity measurements were similar in predicting HTN and DM (AUC: 0.6–0.7). The universal WC threshold of ≥ 35 in. missed 13% of the hypertensive patients and 12% of the diabetic patients. The Asian WC threshold of ≥ 31.5 in. increased detection of HTN and DM but with a high rate of false positives. The traditional BMI ≥ 25 kg/m2 threshold missed 35% of those with hypertension and 24% of those with diabetes. The Asian BMI threshold improved detection but resulted in a high rate of false positives. The suggested WHtR cut-off of ≥ 0.5 missed only 1% of those with HTN and 0% of those with DM. The three obesity measurements had similar but modest ability to predict HTN and DM in FAW. Using Asian-specific thresholds increased accuracy but with a high rate of false positives. Whether FAW, especially at older ages, should be encouraged to reach these lower thresholds needs further investigation because of the high false positive rates.

Objectives-Increasing evidence confirms microbubble (MB) augmented ultrasound (US) thrombolysis enhances clot lysis with or without tissue plasminogen activator (tPA). Intracranial hemorrhage (ICH) is a major complication militating against tPA use in acute ischemic stroke. We quantified the incidence of ICH associated with tPA thrombolysis and MB+US therapy and compared infarct volumes in a rabbit model of acute ischemic stroke.Materials and Methods-Rabbits (n=158) received a 1.0-mm clot angiographically injected into the internal carotid artery causing infarcts. Rabbits were randomized to six test groups including: 1) control (n=50), embolized without therapy, 2) US (n=18), 3) tPA only (n=27), 4) tPA +US (n=22), 5) MB+US (n=27), and 6) tPA+MB+US (n=14). Ultrasound groups received pulsed wave US (1 MHz, 0.8 W/cm 2 ) for 1 hour; rabbits with tPA received intravenous tPA (0.9 mg/kg) over 1 hour. Rabbits with MB received intravenous MB (0.16 mg/kg) given over 30 minutes. Rabbits were sacrificed 24 hours later and infarct volume and incidence, location, and severity of ICH were determined by histology and pathological examination.Results-Percentage of rabbits having ICH outside the infarct area was significantly decreased (P=0.004) for MB+US (19%) rabbits compared with tPA+US (73%), US only (56%), tPA (48%), tPA+MB+US (36%), and control (36%) rabbits. Incidence and severity of ICH within the infarct did not differ (P>0.39). Infarct volume was significantly greater (P=0.002) for rabbits receiving US (0.97±0.17%) than for MB+US (0.20±0.14%), tPA+US (0.15±0.16%), tPA (0.14±0.14%), and tPA+MB+US (0.10±20%) rabbits; these treatments collectively, excluding US only, differed (P=0.03) from control (0.45±0.10%).Conclusions-Treatment with MB+US following embolization decreased the incidence of ICH and efficacy was similar to tPA in reducing infarct volume.

Osteogenesis imperfecta (OI) is a group of genetic disorders characterized by brittle bones that are prone to fracture. Although previous studies in animal models investigated the mechanical properties and material composition of OI bone, little work has been conducted to statistically correlate these parameters to identify key compositional contributors to the impaired bone mechanical behaviors in OI. Further, although increased TGF-β signaling has been demonstrated as a contributing mechanism to the bone pathology in OI models, the relationship between mechanical properties and bone composition after anti-TGF-β treatment in OI has not been studied. Here, we performed follow-up analyses of femurs collected in an earlier study from OI mice with and without anti-TGF-β treatment from both recessive (Crtap ) and dominant (Col1a2 ) OI mouse models and WT mice. Mechanical properties were determined using three-point bending tests and evaluated for statistical correlation with molecular composition in bone tissue assessed by Raman spectroscopy. Statistical regression analysis was conducted to determine significant compositional determinants of mechanical integrity. Interestingly, we found differences in the relationships between bone composition and mechanical properties and in the response to anti-TGF-β treatment. Femurs of both OI models exhibited increased brittleness, which was associated with reduced collagen content and carbonate substitution. In the Col1a2 femurs, reduced hydroxyapatite crystallinity was also found to be associated with increased brittleness, and increased mineral-to-collagen ratio was correlated with increased ultimate strength, elastic modulus, and bone brittleness. In both models of OI, regression analysis demonstrated that collagen content was an important predictor of the increased brittleness. In summary, this work provides new insights into the relationships between bone composition and material properties in models of OI, identifies key bone compositional parameters that correlate with the impaired mechanical integrity of OI bone, and explores the effects of anti-TGF-β treatment on bone-quality parameters in these models. © 2016 American Society for Bone and Mineral Research.