Angiotensin receptor blocker and AT1R transgenic deletion reduce coronary arteriole remodeling in db/db mice We performed pilot studies comparing the effectiveness of angiotensin receptor blockade (candesartan) vs. arteriole cell number in db/db mice. Losartan treatment did not impact incremental elastic modulus. However, losartan improved coronary circulation reserve. Our data suggest that Ang IICAT1R signaling mediates, at least in part, coronary arteriole inward hypertrophic remodeling in T2DM without affecting vascular mechanics, further suggesting that targeting the coronary microvasculature in T2DM may help reduce cardiac ischemic events. knockout (kindly provided by Dr. LM Harrison-Barnard) to generate heterozygous db/dbAT1Ra+/? mice and double homozygous (db/dbAT1Ra?/?) mice [36]. Please note that this colony was lost during Hurricane Katrina, preventing further experimentation. All other experiments were conducted on 12 or 16 week-old male control, non-diabetic heterozygous (Db/db; BKS.Cg-method, with the ribosomal protein transcript Rpl13a serving as the internal control [37] and the average Het value for the aorta serving as the second normalizer. 2.3. Drug treatment Control or db/db mice were administered vehicle water or losartan (3 mg/kg/day) (Sigma, 61188), treated water for 4 weeks, beginning at 12 weeks of age. Water bottles were changed 2 times a week. This dose of losartan was chosen based upon a previous statement that doses 10 mg/kg/day in diabetic mice do not impact blood pressure [48]. 2.4. Blood glucose, insulin and plasma Ang II measurements Prior to end point experiments, fasting blood glucose (8C10 hour food withdrawal during the light cycle) was measured from tail vein blood using the Accu-Chek Advantage meter (Roche, Indianapolis, Indiana). Insulin levels were measured using an ELISA kit from Mercodia (Winston-Salem, NC). The provided protocol was followed exactly, with the following exception: All db/db samples were diluted 1:3 with Calibrator 0 answer prior to assay (i.e. 20 L plasma + 40 L Calibrator 0). This was done in anticipation of db/db mouse insulin levels being very high. Samples were read on the SpectraMax M5 spectrophotometer. Plasma Ang II concentrations were measured by radioimmunoassay at Hypertension Core lab at Wake Forest University or college. 2.5. Coronary arteriole isolation and measurement of structural and passive mechanical properties At the end of treatment (16 weeks) mice were anesthetized using 3% isoflurane, vaporized with 100% oxygen. The heart was excised and dissected in chilly physiologic salt answer (PSS). The right ventricle was removed and septal coronary arterioles ( 120 m internal diameter) at the level of the superior papillary muscle were isolated, excised and mounted onto two glass microcannulas within a pressure myograph chamber (Living Systems, Burlington, VT) as previously explained by our lab [19]. One vessel was isolated per animal. Prior to any measurements, vessels were equilibrated for 30 min under constant intraluminal pressure (50 mm Hg) at 37 C in PSS. Internal diameter and left and right wall thickness (WT) were continuously monitored by a video image analyzer (Living Systems) and recorded using LabCart 6 data acquisition software connected to a PowerLab 16/30 (ADInstruments, Inc., Colorado Springs, CO). All experiments were Furilazole performed in Ca2+-free PSS in the presence of 2 mM EGTA and 100 M sodium nitroprusside. A passive pressureCdiameter curve was generated by increasing intraluminal pressure from 0 to 125 mm Hg. Coronary wall thickness (WT) and internal diameters (Di) were recorded at each pressure. The following structural and mechanical parameters were calculated as previously explained [19]: External diameter (De) = is the internal coronary diameter (in mm) measured in B-mode ultrasound images, VTI is the velocityCtime-integral (in mm), or area under the curve of the Doppler blood Furilazole flow velocity tracing, and HR is the heart rate. Coronary flow reserve (CFR) = CBFhyperemia/CBFbaseline where CBFhyperemia is coronary flow measured during 3% isoflurane administration. 2.8. Blood pressure telemetry Radiotelemetry transmitters (PA-C10, Data Sciences, St. Paul, MN) were implanted into mice as described by our lab [19]. Briefly, mice were anesthetized with 2% isoflurane, and the right carotid artery was isolated and cannulated with a telemeter catheter connected to a radio-telemetry transmitter. Since db/db mice are more sensitive to surgical stressors, data recording commenced after the return of normal diurnal blood pressure rhythms (7C10 days). Data were collected for 10 s every 15 Furilazole min for a total of 4 weeks using DataQuest A.R.T. 4.2 software. 2.9. Elastin immunofluorescence Paraffin-embedded heart sections (5 m.Water bottles were changed 2 times a week. treatment did not affect incremental elastic modulus. However, losartan improved coronary flow reserve. Our data suggest that Ang IICAT1R signaling mediates, at least in part, coronary arteriole inward hypertrophic remodeling in T2DM without affecting vascular mechanics, further suggesting that targeting the coronary microvasculature in T2DM may help reduce cardiac ischemic events. knockout (kindly provided by Dr. LM Harrison-Barnard) to generate heterozygous db/dbAT1Ra+/? mice and double homozygous (db/dbAT1Ra?/?) mice [36]. Please note that this colony was lost during Hurricane Katrina, preventing further experimentation. All other experiments were conducted on 12 or 16 week-old male control, non-diabetic heterozygous (Db/db; BKS.Cg-method, with the ribosomal protein transcript Rpl13a serving as the internal control [37] and the average Het value for the aorta serving as the second normalizer. 2.3. Drug treatment Control or db/db mice were administered vehicle water or losartan (3 mg/kg/day) (Sigma, 61188), treated water for 4 weeks, beginning at 12 weeks of age. Water bottles were changed 2 times a week. This dose of losartan was chosen based upon a previous report that doses 10 mg/kg/day in diabetic mice do not affect blood pressure [48]. 2.4. Blood glucose, insulin and plasma Ang II measurements Prior to end point experiments, fasting blood glucose (8C10 hour food withdrawal during the light cycle) was measured from tail vein blood using the Accu-Chek Advantage meter (Roche, Indianapolis, Indiana). Insulin levels were measured using an ELISA kit from Mercodia (Winston-Salem, NC). The provided protocol was followed exactly, with the following exception: All db/db samples were diluted 1:3 with Calibrator 0 solution prior to assay (i.e. 20 L plasma + 40 L Calibrator 0). This was done in anticipation of db/db mouse insulin levels being very high. Samples were read on the SpectraMax M5 spectrophotometer. Plasma Ang II concentrations were measured by radioimmunoassay at Hypertension Core lab at Wake Forest University. 2.5. Coronary arteriole isolation and measurement of structural and passive mechanical properties At the end of treatment (16 weeks) mice were anesthetized using 3% isoflurane, vaporized with 100% oxygen. The heart was excised and dissected in cold physiologic salt solution (PSS). The right ventricle was removed and septal coronary arterioles ( 120 m internal diameter) at the level of the superior papillary muscle were isolated, excised and mounted onto two glass microcannulas within a pressure myograph chamber (Living Systems, Burlington, VT) as previously described by our lab [19]. One vessel was isolated per animal. Prior to any measurements, vessels were equilibrated for 30 min under constant intraluminal pressure (50 mm Hg) at 37 C in PSS. Internal diameter and left and right wall thickness (WT) were continuously monitored by a video image analyzer (Living Systems) and recorded using LabCart 6 data acquisition software connected to a PowerLab 16/30 (ADInstruments, Inc., Colorado Springs, CO). All experiments were performed in Ca2+-free PSS in the presence of 2 mM EGTA and 100 M sodium nitroprusside. A passive pressureCdiameter curve was generated by increasing intraluminal pressure from 0 to 125 mm Hg. Coronary wall thickness (WT) and internal diameters (Di) were recorded at each pressure. The following structural and mechanical parameters were calculated as previously described [19]: External diameter (De) = is the internal coronary diameter (in mm) measured in B-mode ultrasound images, VTI is the velocityCtime-integral (in mm), or area under the curve of the Doppler blood flow velocity tracing, and HR is the heart rate. Coronary flow reserve (CFR) = CBFhyperemia/CBFbaseline where CBFhyperemia is coronary flow measured during 3% isoflurane administration. 2.8. Blood pressure telemetry Radiotelemetry transmitters (PA-C10, Data Sciences, St. Paul, MN) were implanted into mice as described by our lab [19]. Briefly, mice were anesthetized with 2% isoflurane, and the right carotid artery was isolated and cannulated with a telemeter catheter connected to a radio-telemetry transmitter. Since db/db mice are more sensitive to surgical stressors, data recording commenced after the return of normal diurnal blood pressure rhythms (7C10 days). Data were collected for 10 s every 15 min for a total of 4 weeks using DataQuest A.R.T. 4.2 software. 2.9. Elastin immunofluorescence Paraffin-embedded heart sections (5 m thick) from 16-week animals were incubated with an elastin Furilazole antibody (1:75,.Immunofluorescence staining revealed increased elastin in db/db mice compared to controls (Fig. in db/db mice. Losartan treatment did not affect incremental elastic modulus. However, losartan improved coronary flow reserve. Our data suggest that Ang IICAT1R signaling mediates, at least in part, coronary arteriole inward hypertrophic remodeling in T2DM without affecting vascular mechanics, further suggesting that targeting the coronary microvasculature in T2DM may help reduce cardiac ischemic events. knockout (kindly provided by Dr. LM Harrison-Barnard) to generate heterozygous db/dbAT1Ra+/? mice and double homozygous (db/dbAT1Ra?/?) mice [36]. Please note that this colony was lost during Hurricane Katrina, preventing further experimentation. All other experiments were conducted on 12 or 16 week-old male control, non-diabetic heterozygous (Db/db; BKS.Cg-method, with the ribosomal protein transcript Rpl13a serving as the internal control [37] and the average Het value for the aorta serving as the second normalizer. 2.3. Drug treatment Control or db/db mice were administered vehicle water or losartan (3 mg/kg/day time) (Sigma, 61188), treated water for 4 weeks, beginning at 12 weeks of age. Water bottles were changed 2 times a week. This dose of losartan was chosen based upon a previous statement that doses 10 mg/kg/day time in diabetic mice do not impact blood pressure [48]. 2.4. Blood glucose, insulin and plasma Ang II measurements Prior to end point experiments, fasting blood glucose (8C10 hour food withdrawal during the light cycle) was measured from tail vein blood using the Accu-Chek Advantage meter (Roche, Indianapolis, Indiana). Insulin levels were measured using an ELISA kit from Mercodia (Winston-Salem, NC). The offered protocol was adopted exactly, with the following exclusion: All db/db samples were diluted 1:3 with Calibrator 0 remedy prior to assay (i.e. 20 L plasma + 40 L Calibrator 0). This was done in anticipation of db/db mouse insulin levels being very high. Samples were read on the SpectraMax M5 spectrophotometer. Plasma Ang II concentrations were measured by radioimmunoassay at Hypertension Core lab at Wake Forest University or college. 2.5. Coronary arteriole isolation and measurement of structural and passive mechanical properties At the ARVD end of treatment (16 weeks) mice were anesthetized using 3% isoflurane, vaporized with 100% oxygen. The heart was excised and dissected in chilly physiologic salt remedy (PSS). The right ventricle was eliminated and septal coronary arterioles ( 120 m internal diameter) at the level of the superior papillary muscle were isolated, excised and mounted onto two glass microcannulas within a pressure myograph chamber (Living Systems, Burlington, VT) as previously explained by our lab [19]. One vessel was isolated per animal. Prior to any measurements, vessels were equilibrated for 30 min under constant intraluminal pressure (50 mm Hg) at 37 C in PSS. Internal diameter and remaining and right wall thickness (WT) were continuously monitored by a video image analyzer (Living Systems) and recorded using LabCart 6 data acquisition software connected to a PowerLab 16/30 (ADInstruments, Inc., Colorado Springs, CO). All experiments were performed in Ca2+-free PSS in the presence of 2 mM EGTA and 100 M sodium nitroprusside. A passive pressureCdiameter curve was generated by increasing intraluminal pressure from 0 to 125 mm Hg. Coronary wall thickness (WT) and internal diameters (Di) were recorded at each pressure. The following structural and mechanical parameters were determined as previously explained [19]: External diameter (De) = is the internal coronary diameter (in mm) measured in B-mode ultrasound images, VTI is the velocityCtime-integral (in mm), or area under the curve of the Doppler blood flow velocity tracing, and HR is the heart rate. Coronary circulation reserve (CFR) = CBFhyperemia/CBFbaseline where CBFhyperemia is definitely coronary flow measured during 3% isoflurane administration. 2.8. Blood pressure telemetry Radiotelemetry transmitters (PA-C10, Data Sciences, St. Paul, MN) were implanted into mice as explained by our lab [19]. Briefly, mice were anesthetized with 2% isoflurane, and the right carotid artery was.