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Introduction Adenosine triphosphate (ATP) is postulated to be a major player in the local control of skeletal muscle vascular tone during exercise. Exercise during hypoxic conditions significantly augments skeletal muscle blood flow and elevates venous plasma levels of ATP compared to normoxic conditions in young healthy adults. This suggests that the elevated levels of ATP during hypoxic exercise contribute to the augmented vasodilation. Nitric oxide synthase (NOS) inhibition has also been shown to blunt the effects of hypoxia on exercise vasodilation. Therefore, the aim of the current study was to evaluate the effect of NOS inhibition and hypoxia on vasodilator responses and plasma ATP levels during exercise in healthy young adults. Methods 10 young volunteers (age = 28 ± 1 years, 6 men/4 women) performed four minutes of forearm handgrip exercise at 20% of their maximal voluntary contraction (MVC). The forearm handgrip exercise was performed under normoxia and hypoxia (SpO2 = ~80%) with and without NOS inhibitor, LNMMA administered via a brachial artery catheter. Beat to beat blood pressure and heart rate were recorded. Forearm blood flow was measured using Doppler ultrasound and normalized for arterial pressure to determine forearm vascular conductance (FVC). Venous blood was obtained from a retrograde intravenous catheter at rest and during the last minute of each exercise bout. Plasma ATP was measured using the luciferin‐luciferase technique. Results During saline and normoxia FVC was 43.4 ± 8.4 at rest and 307.8 ± 22.6 ml/min/100mmHg during exercise. With hypoxia FVC increased to 59.35 ± 10.5 at rest and 370.9 ± 33.3 ml/min/100mmHg during exercise (both p<0.05 vs. normoxia). During LNMMA and normoxia FVC did not change significantly compared to saline 33.9 ± 4.4 at rest and 283.5 ± 23.2 ml/min/100mmHg during exercise. With hypoxia and LNMMA FVC was 35.04 ± 4.1 at rest (p>0.05 vs. normoxia) and 326.0 ± 23.9 ml/min/100mmHg during exercise (p<0.05 vs. normoxia). When changes from baseline are evaluated, hypoxia augmented the vasodilator responses exercise to exercise during both saline and LNMMA. ATP was higher during 20% MVC exercise with saline during hypoxia as compared to normoxia (65.6 ± 12.1 vs. 108.1 ± 23.2 versus nmol/L, p=0.058). It was also higher during 20% MVC exercise with LNMMA (81.7 ± 9.5 vs. 106.1 ± 14.7 nmol/L, p=0.055). When changes from baseline are evaluated, hypoxia augmented venous ATP concentration with exercise during both saline and LNMMA. Conclusions Our results suggest that increased endogenous ATP release during hypoxic exercise continued to evoke vasodilation during NOS inhibition. A likely source of this ATP is from red blood cells and this interpretation is consistent with findings demonstrating that NOS inhibition has limited effects on ATP mediated dilation in the human forearm. Support or Funding Information This research was supported by National Institutes of Health Research Grants HL‐119337, CTSA UL1 TR000135 and the Caywood Professorship via the Mayo Foundation

Background Nitric oxide (NO) contributes ~30% to the rate of vasodilation during dynamic exercise in healthy humans. This NO‐mediated dilation occurs via increases in cyclic guanosine monophosphate (cGMP), which is catabolized by phosphodiesterase‐5 (PDE‐5). Therefore, we sought to determine the nature of NO‐cGMP signalling to the exercise vasodilator response in healthy humans. We hypothesized the time‐course of exercise vasodilation would be: 1) accelerated by sildenafil citrate (SDF, PDE‐5 inhibitor) 2) slowed by the NO synthase inhibitor L‐N G ‐monomethyl arginine (L‐NMMA) 3) similarly slowed by the combination of SDF+L‐NMMA. Methods Data from 29 healthy individuals were included in the present study. All subjects completed 5‐min of dynamic forearm exercise at 15% of maximal voluntary contraction under control conditions (CON, arterial saline infusion), followed by an identical exercise protocol during: 1) PDE‐5 inhibition via SDF (100mg oral dose, n = 8), 2) NOS inhibition via intra‐arterial infusion of L‐NMMA (50 mg loading dose and 1 mg/min maintenance dose, n = 15), or 3) combined SDF+L‐NMMA (n = 6). Forearm blood flow (FBF; Doppler ultrasound) and mean brachial artery blood pressure (MAP, transducer) were measured continuously. To determine time to steady‐state vasodilation, forearm vascular conductance (FVC = FBF/100mmHg/dL FAV) data were curve fit with a monoexponential model and mean response time (MRT, time to achieve 63% of steady state response) was calculated. In addition, the increase in relative forearm vascular conductance from rest to steady state exercise (average of the last 30s of exercise, ΔFVC = FVC exercise − FVC rest ) was used to assess the total vasodilatory response. Results Data are reported as mean ± SE (FVC = mL/dL/100mmHg/min, MRT = s) . In contrast to our hypothesis, SDF alone had no effect on exercise vasodilation (Δ FVC CON = 20±3 vs. SDF = 21±3, p = 0.50) or time to steady‐state vasodilation (MRT: CON = 32± 7 vs SDF= 33±4, p = 0.90) in young healthy adults. NOS inhibition with L‐NMMA reduced ΔFVC (CON = 16±2 vs. L‐NNMA = 14±1, p = 0.049) and increased the time to reach steady‐state vasodilation (MRT: CON = 22±5 vs. L‐NMMA = 40±6, p = 0.01). Surprisingly, when subjects were given L‐NMMA combined with SDF, reductions in Δ FVC or increases in MRT were no longer observed (Δ FVC: CON = 23±4 vs. SDF+L‐NMMA = 23±6, p = 0.90; MRT: CON = 45±8 vs. SDF+L‐NMMA = 49±11, p = 0.80). Conclusion These data indicate inhibiting PDE‐5 during moderate exercise does not improve the magnitude or time‐course of vasodilation. Whereas, NOS inhibition limits the magnitude and time‐course of the vasodilatory response to dynamic handgrip exercise in healthy adults, combined inhibition of PDE‐5 restores the response to control levels. These data suggest PDE‐5 inhibition does not potentiate the NO‐cGMP pathway during moderate exercise. However, PDE‐5 inhibition may have therapeutic use in pathologies where limited NO bioavailability may contribute to impaired blood flow responses to exercise. Support or Funding Information American Heart Association (15POST23100020) ‐ JMKNational Institutes of Health (HL46493) ‐ MJJ National Institutes of Health (HL078019) ‐ MJJ National Institutes of Health (HL105820) ‐ WGS National Institutes of Health (RR17520) ‐ N/A National Institutes of Health (UL1 TR000135) ‐ N/A

Red blood cells (RBCs) release ATP in direct proportion to the degree of hemoglobin (Hb) deoxygenation, which binds to purinergic receptors on the endothelium and stimulates vasodilation. Accordingly, RBCs act as both a ‘sensor’ for oxygen demand and an ‘effector’ for increasing oxygen delivery to facilitate the matching of tissue oxygen supply and demand. We have recently demonstrated that Hb deoxygenation‐induced ATP release is impaired in RBCs from healthy older vs. young adults, but the underlying mechanisms are not fully understood. Cyclic AMP (cAMP) has been shown to be an important component of the signaling cascade for RBC ATP release, and decreased cAMP signaling contributes to impaired ATP release from RBCs of humans with type 2 diabetes. As such, treatment with cilostazol to inhibit phosphodiesterase 3 (PDE3) hydrolysis of cAMP improves Hb deoxygenation‐induced ATP release from RBCs of these patients. Whether similar alterations in cAMP signaling contribute to impaired RBC ATP release in healthy older adults is unclear. Thus, in the present study we tested the hypothesis that PDE3 inhibition would restore Hb deoxygenation‐induced ATP release from RBCs of healthy older adults. Isolated RBC ATP release in response to normoxic (PO 2 ~111 mmHg) and hypoxic (PO 2 ~26 mmHg) stimuli was quantified in RBCs from young (25 ± 1 years; n = 4) and older (65 ± 3 years; n = 6) adults using the luciferin‐luciferase technique following RBC incubation with either dimethylformamide (DMF; vehicle control) or 100 μM cilostazol (PDE3 inhibitor). The relative change in ATP release from normoxia to hypoxia in control conditions was impaired in RBCs from older (56.1 ± 11.0%) vs. young (129.1 ± 16.1%) adults (p < 0.05). Cilostazol resulted in a slight, but non‐significant improvement in RBC ATP release in older adults (74.2 ± 15.0%; p = 0.32 vs. control), whereas there was no effect on RBC ATP release in young adults (123.4 ± 37.5%; p = 0.84 vs. control). RBC intracellular ATP was not different between age groups or treatments (p > 0.05). These preliminary data suggest that greater cAMP hydrolysis via PDE3 in response to Hb deoxygenation is not a primary contributor to impaired RBC ATP release in older adults. Support or Funding Information HL119337, F31HL126377

Doxorubicin (Dox), an anthracycline‐based chemotherapeutic agent, is highly effective at reducing recurrence and mortality in breast cancer patients. However, this anti‐cancer drug elicits dose‐dependent cardiotoxicity. Multiple mechanisms have been implicated in its pathogenesis, one of which involves the development and progression of inflammation mediated by activation of the TLR4/NFkappaB pathway. However, we recently demonstrated that chronic in vivo Dox treatment reduced expression of the NFkappaB downstream proinflammatory mediator, cyclooxygenase‐2 (COX‐2), in cerebral and peripheral vasculatures isolated from ovariectomized female rats. Therefore, in the current study we tested whether Dox similarly alters vascular proinflammatory enzyme expression in the human vasculature and whether this response is dose dependent. We further investigated whether Dox alters upstream mediators of COX‐2 expression by assessing levels of expression and activation of NFkappaB and an additional downstream gene product, TLR4. Primary human coronary vascular smooth muscle cells of female origin were studied at passage 6 and treated with vehicle or Dox (0.3, 1.0, and 5 μM). Following drug treatment, cells were isolated, homogenized, and lysates (whole cell; nuclear and cytosolic) analyzed for NFkappaBp65, COX‐2, and TLR4 protein levels via immunoblotting. Our results demonstrate that while total NFkappaB levels were unaffected following Dox, enhanced translocation of NFkappaB to the nucleus was observed. However, even in the presence of Dox‐induced NFkappa translocation, basal expression of its downstream mediator, COX‐2, was dose dependently attenuated. Similar to COX‐2, a trend for a decrease in TLR4 levels was observed. Dox had no effect on cell morphology or density, as visualized via light microscopy. In conclusion, although others have suggested the involvement of NFkappaB activation during the development and progression of Dox‐induced inflammation in cardiac muscle, our studies demonstrate a possible novel action for the anticancer agent targeting important downstream mediators of inflammation in the female vasculature. Future studies will evaluate the impact of Dox on COX‐2 at the transcriptional or translational level to determine the mode of molecular regulation. Support or Funding Information University of Arizona Sarver Heart Center (RG and MS); Mayo Clinic Dept. of Medicine Extramural Chair Award in collaboration with Arizona State University (SA)

The bone marrow microenvironment acts as a protective niche for malignant cells. Within the bone marrow microenvironment is the endosteal niche, which is the area where the ossified surface of the bone and marrow cavity interact. The endosteal niche provides the opportunity for hematological malignancies such as acute myeloid leukemia (AML) of the bone marrow to interact with osteoblasts from the bone. To study the interaction between AML cells and osteoblasts, our lab developed a co‐culture model of osteoblasts (MC3T3 osteoblast cell line) and AML cells (KG1a cell line or patient samples from bone marrow aspirates). AML cells expressing high levels of CXCR4 were cultured alone or with osteoblasts and then challenged with SDF‐1, the ligand to CXCR4 and a chemokine readily abundant in the bone marrow microenvironment that our lab has previously shown actually induces apoptosis in AML cells expressing high levels of CXCR4 and that osteoblasts are able to protect AML cells from SDF‐1 induced apoptosis. Our lab now shows that treating osteoblasts with cyclosporine (CSA) prevents their ability to effectively protect AML cells from SDF‐1 induced cell death as assayed by annexin‐V staining of AML cells by flow cytometry. CSA treatment of osteoblasts decreased protein levels of tissue non‐specific alkaline phosphatase (ALPL), a phosphatase necessary for osteoblast mineralization, compared to vehicle control. As such, osteoblasts were treated with control siRNA or ALPL siRNA. CXCR4 expressing AML cells were then co‐cultured with the transfected osteoblasts and challenged with SDF‐1. siRNA knockdown of ALPL in osteoblasts was sufficient to prevent protection of AML cells from the apoptotic stimuli. These studies indicate that osteoblast differentiation plays a role in mediating the protection of AML cells. Therefore, targeting specific differentiation factors in osteoblasts may help to make to bone marrow microenvironment a more hostile environment for AML cells and aid in better malignancy eradication. Support or Funding Information This study was supported by NIH R21 CA194217 (to K.E.H.). R.M.S. is supported by the Mayo Clinic Medical Scientist Training Program Robert L. Howell Physician‐Scientist Scholarship.

Relief of aversive states, including pain, is rewarding and associated with an increase in dopamine (DA) release in the mesolimbic reward circuit. Measurement of DA in the nucleus accumbens (NAc) is usually accomplished with microdialysis but this method does not allow determination of the temporal relationship to pain, and to its relief. In this study, we used isoflurane anesthetized rats to measure NAc DA release on a millisecond to second timescale using fast scan cyclic voltammetry (FSCV). Our approach was to standardize the depth of anesthesia by a reflexive pain response to noxious heat, the tail‐flick test. The depth of anesthesia was standardized to this biological response of approximately 5.0 sec latency. FSCV involves the application of a specific triangular waveform at a high scan rate, allowing for oxidation and reduction of dopamine detected by a specialized electrode previously implanted in the NAc shell. Application of noxious heat to the tail was accompanied by a decrease in the DA signal that peaked at the time of the tail‐flick. Following the tail‐flick, there was a rebound increase in DA signal that peaked after 2.9 s. The data show a time‐locked DA response to pain onset, and to pain offset, likely related to the negative and positive components of pain and its relief. Support or Funding Information Supported by DA034975 from the NIH

Radiology is often incorporated into medical school anatomy courses. Radiology education furthers understanding of anatomical relationships and prepares students to apply anatomical knowledge in clinical practice. The aim of this study was to investigate how cognitive performance on the radiology components (RC) of a first‐year medical school anatomy course are related to performance on non‐radiological components (NRC). We hypothesized that there is no correlation between these two performance variables, because they rely on different cognitive skills. Thus, anatomy education alone will not prepare students for the interpretation of normal anatomy on radiological images. Medical students (n=51) enrolled in a first‐year anatomy course at Mayo Clinic School of Medicine were included in this study. Assessment data from three lab practical examinations were collected, and RC and NRC questions were compared. A bivariate linear regression and statistical tests were conducted. A subgroup analysis was used to compare students whose RC performance was greater than their NRC performance (RC>NRC, n=33) to students whose RC performance was less than their NRC performance (RC<NRC, n=18). When considering all three exams, students correctly answered 71.1% of the RC questions and 68.3% of NRC questions (p=0.047). Bivariate linear regression demonstrated a moderate positive correlation between RC and NRC performance (p<0.01, r= 0.51); however, performance on RC questions only accounted for 24% of the variability of NRC scores. Additionally, students in the RC>NRC group had significantly higher scores on RC questions than the students in the RC<NRC group (74% vs 66%, p<0.02). Conversely, students in the RC>NRC group had significantly lower scores on NRC questions than the students in the RC<NRC group (61% vs 74%, p=0.001). These results indicate that while RC performance has some influence on NRC performance it does not account for the majority of the difference observed. Such results suggest that the cognitive skills involved in both radiology and anatomy education are different. Therefore, dedicated radiology education is an important component of an anatomy course as it fosters the development of novel skills that are necessary when interpreting normal anatomy on radiological images in a clinical setting. Support or Funding Information None

Renal artery stenosis (RAS) is a common public health problem, affecting up to 7% of individuals over 65 years of age. Optimal therapy for patients with RAS is controversial, as recent studies have indicated that revascularization via stenting fails to improve renal function or patient survival. Medical therapy is associated with high morbidity and mortality, primarily due to cardiovascular disease. However, morphologic correlates for reversibility following restoration of blood flow have not been adequately defined. The purpose of this study was to standardize an experimental model for the restoration of blood flow, employing a well characterized murine RAS model. Total N=10 C57B/6J male mice were used. RAS was induced on the right renal artery by putting a polytetrafluoroethylene cuff (N=6) and sham surgery was performed without cuff placement (N=4). To determine the feasibly of restoring the blood flow to the stenotic kidney, cuff was taken off at day 7 following the RAS surgery. Blood pressure measurements were done using tail cuff method at baseline, before taking cuff off and then every week until harvest. MRI images were captured using an Avance DRX 700WB spectrometer with a vertical 16.4 T wide bore magnet connected with the imaging tools at baseline, 3 days after putting the cuff on or sham surgery, and at 3 weeks after cuff off. The animals were harvested at day 28 following surgery and kidney weights are obtained. Baseline renal perfusion (1018±41.6 ml/100g/min) was significantly reduced (634.7±141.3 ml/100g/min, p=0.01) following cuff placement. Perfusion was restored to normal (955±59.8 ml/100g/min) at 28 days following cuff removal at 7 days after RAS. Similarly, volume of the stenotic kidney was significantly reduced (at day 3 after RAS 172±8.6 mm 3 ) compared to baseline (206.3±4.4 mm 3 , p=0.0007). There was no significant difference in perfusion and volume after cuff off compared to baseline and sham groups. There was no significant difference in kidney weight between cuff‐off (167± 4.7 mg) and sham groups (163±9.95 mg). Also, there was no difference in heart weight between cuff off (150±3.7mg) and sham groups (148±6.7mg). We conclude that this model will provide the basis for determining the latest time point associated with recovery of renal structure and potential histopathologic markers that predict preservation of renal structure following cuff removal. Also, this model would provide the basis for determining efficacy of additional therapies to target inflammation, etc. to widen the “therapeutic window” during which restoration of renal blood flow is beneficial. Support or Funding Information This project is funded by National Institutes of health, NIAID R01 AI100911.

Background A multitude of studies have suggested that there is a link between obesity and inflammation. Various in vitro and animal studies have shown that saturated fatty acids are largely responsible for the induction of various pro‐inflammatory pathways. As vegetarians consume about half the amount of saturated fat as omnivores, we hypothesized that they would have less inflammation for any given amount of obesity. Objective To quantitate the inflammatory status of adipose tissue from obese vegetarians and omnivores. Methods The study included 8 obese vegetarians (1 male) and 8 obese omnivores matched for age, sex, and BMI. Body composition was measured by DXA. A blood sample was collected to measure plasma free fatty acids (FFA), subcutaneous adipose tissue biopsies to measure macrophage content and muscle biopsies to quantify diacylglycerol (DAG) and ceramide content. Each participant also completed a Food Frequency Questionnaire (FFQ) to determine their average dietary intake. Results By design there were no differences in age, weight, BMI, percentage body fat, fat free mass or adipocyte size between the omnivore and vegetarian group. We found that vegetarians consume lower amounts of saturated fatty acids and cholesterol while consuming higher amounts of fiber and vitamin C than the omnivore participants ( p = 0.06, 0.04, 0.004, 0.04 respectively). The plasma FFA of vegetarians revealed that they had greater proportions of the polyunsaturated fatty acids linoleic, linolenic ( p = 0.01, 0.02 respectively) as well as increased monounsaturated oleic acid ( p = 0.04). The inflammatory status of adipose tissue was quantified by measuring macrophage content (total, pro‐inflammatory M1, and anti‐inflammatory M2 macrophages). The vegetarian group had significantly lower adipose tissue total ( p = 0.004), M1 ( p = 0.002), and M2 ( p = 0.01) macrophage burden when compared the omnivore group. There was a difference, however, in muscle DAG and ceramide content between the two groups. Conclusions As expected, vegetarians consume a diet that varies in key macro‐ and micronutrients that may affect inflammatory status. Indeed, the vegetarian group had significantly lower adipose tissue macrophage burden than the omnivore participants despite their obese state. While these variations in dietary intake seem to have a positive impact on the adipose tissue immune cell milieu there was no difference in the potential inflammatory cell signaling components of the muscle tissue, DAGs and ceramides, between the two groups. These promising results display the importance of dietary intake in modulating adipose tissue inflammation and potentially chronic disease.

Ovarian cancer remains a devastating disease with 5 year survival rate in advanced stage of only 27%. Advanced stage tumors metastasize through continuous shedding of tumor cells into the peritoneal fluid, aided by the resistance of these cells to anoikis, a form of apoptosis normally triggered by loss of cell‐matrix interactions. We have previously shown that the serine protease inhibitor SPINK1 activates anoikis resistance in ovarian cancer cells via its protease inhibitory function; however, the specific relevant protease target(s) remain unknown. To find these targets, we have synthesized an activity‐based protein probe with a diphenylphosphonate warhead that covalently modifies serine proteases, with greatest specificity towards trypsin‐like proteases. We have used this probe to identify ovarian cancer cell secreted and membrane‐bound serine proteases possessing differential activity in the presence versus absence of SPINK1, using a proteomics approach. We are evaluating candidate proteases for involvement in anoikis resistance in ovarian cancer models. Our studies implicate a serine protease network in regulation of anoikis in ovarian cancer cells. By identifying critical proteases that control anchorage‐independent viability in ovarian cancer metastasis, we aim to elucidate important downstream pathways and ultimately develop therapeutic approaches that can intervene in mechanisms of anoikis resistance. Support or Funding Information Mayo Clinic Graduate School, Rochester MN