Functional assessment of cryopreserved human femoral arteries for pharmaceutical research

An established method for cryopreservation that might preserve the vascular and endothelial responses of human femoral arteries (HFAs) to be transplanted as allografts was studied. HFAs were harvested from multiorgan donors and stored at 4 degrees C in saline solution before cryostorage. Thirty HFA rings were isolated and randomly assigned to one control group of unfrozen HFAs (eight rings) and one group of cryopreserved HFAs (22 rings).Cryopreservation was performed in RPMI solution containing dimethylsulfoxide (DMSO) and the rate of cooling was -1 degrees C/min until -40 degrees C and faster rates until -150 degrees C was reached. The contractile and relaxant responses of unfrozen and frozen/thawed arteries were assessed in organ bath by measurement of isometric force generated by the HFAs.After thawing, the maximal contractile responses to the contracting agonist tested (noradrenaline) were in the range of 43% of the responses in unfrozen HFAs. The endothelium-independent responses to sodium nitroprusside were not altered whereas the endothelium-dependent relaxant responses to acetylcholine were weakly altered.The cryopreservation method used provided a limited preservation of contractility of HFAs, a good preservation of the endothelium-independent relaxant responses, and a good preservation of endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol, such as a slower rate of cooling and a more controlled stepwise addition of DMSO, might allow better post-thaw functional recovery.     

Freezing without surrounding cryomedium preserves the endothelium and its function in human internal mammary arteries

PURPOSE: Cryopreserved human blood vessels may become important tools in bypass surgery. Optimal cryopreservation of an arterial graft should, therefore, preserve both histological and physiological characteristics of smooth muscle and endothelium comparable to the unfrozen artery. METHODS: Rings from human internal mammary arteries (IMA) were investigated in vitro either unfrozen or after immersion into a cryomedium (RPMI 1640 containing 1.8M Me2SO and 0.1M sucrose) and cryostorage with and without surrounding medium. RESULTS: In unfrozen IMA, neither contractile responses to noradrenaline (NA) nor endothelium-dependent relaxant responses to acetylcholine (ACH) was modified after exposure of the IMA to cryomedium or during activation of protein kinase C by phorbol-12,13-dibutyrate (PDBu). Exposure to cryomedium with gradually increasing Me2SO content before starting the cooling process did not improve the post-thaw functional activity of the artery. Optimal post-thaw recovery of contractile responses to NA and PGF(2alpha) was observed after freezing at a speed of -1.2 and -3 degrees C/min in arteries stored with and without surrounding cryomedium. Compared to unfrozen controls, the ACH-induced endothelium-dependent relaxation during active tone induced by 3 microM PGF(2alpha) reached 16 and 56% after freezing with and without surrounding medium. All functional data were reflected by electron microscopy images showing considerably better preservation of the endothelial layer after freezing without medium. CONCLUSION: Freezing of human arteries at a mean cooling rate of -3 degrees C/min and storage without surrounding medium offers the prospect of optimal preservation of both smooth muscle and endothelial function in cryopreserved human IMA.     

Functional assessment of cryopreserved pig aortas for pharmaceutical research

Several in vitro studies have demonstrated diminished post-thaw functional activity. Therefore, the aim of this study was to investigate the consequences of thawing and storage method used on the post-thaw functional activity of cryopreserved pig aortas with the aim of adjusting the freezing and thawing protocol so that the vascular segments are preserved in the best possible state, maintaining structure and functionality so that they can later be transplanted with success. In vitro responses of frozen, thawed pig aortas were used to investigate the functional activity after thawing at 15 degrees C and 100 degrees C/min and after storage in gas or liquid phase of liquid nitrogen. Cryopreservation was performed in RPMI 1640 medium + 10% dimethylsulfoxide and the rate of cooling was -1 degrees C/min, until -150 degrees C was reached.After thawing the maximal contractile responses to all the contracting agonists tested (KCl, noradrenaline) were in the ranges of 13-27% compared with the responses in unfrozen pig aortas. Contractile responses were slightly better when thawing was performed at 15 degrees C/min compared with 100 degrees C/min. The endothelium independent relaxant responses to sodium nitroprusside were reduced ( P < 0.05). Cryostorage of pig arteries also resulted in a loss of the endothelium-dependent relaxant response to acetylcholine. The cryopreservation method used provided a limited preservation of pig aorta contractibility, a reduction of the endothelium independent relaxant responses, and no apparent preservation of the endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol might allow better post-thaw functional recovery of pig aortas.     

Retention of endothelium-dependent vasodilatory responses in canine coronary arteries following cryopreservation

In the present study, the cryoprotective effect of dimethyl sulfoxide (Me2SO) and fetal calf serum (FCS) on coronary endothelium and endothelium-dependent relaxation (EDR) responses was studied in isolated canine coronary arteries following cryostorage at -75 degrees C. Compared to the freshly isolated coronary arteries, the EDR responses to acetylcholine, thrombin, and calcium ionophore were not significantly altered following 1 day storage at -75 degrees C in the presence of 1.8 M Me2SO and 20% FCS. Prolonged cold storage to 7 days, however, resulted in a slight, but significant, rightward shift of the concentration-response curves of acetylcholine and thrombin, but not calcium ionophore. The maximum relaxant response after 7-day cryostorage was 80 to 85% of fresh controls. Omission of FCS from the cryostorage incubation medium further accentuated the loss of EDR responses to all three endothelium-dependent vasodilators tested. Scanning electron microscopic examinations of the intimal surface of the Me2SO and FCS cryostored canine coronary arteries confirmed the preservation of intimal endothelial cells following 1 or 7 days of storage at -75 degrees C, while significant patches of loss of endothelial cells were observed in the arteries cryostored only in the presence of Me2SO. No significant inhibitory effect of cryostorage was observed for the direct, endothelium-independent relaxation induced by isoproterenol, regardless of the presence or absence of FCS. These results demonstrate that slow freezing of canine coronary arteries to -75 degrees C in Krebs-Henseleit solution containing Me2SO and FCS provides good preservation of the vascular smooth muscle function and endothelium-dependent vasodilatory responses.     

Exercise training improves femoral artery blood flow responses to endothelium-dependent dilators in hypercholesterolemic pigs

We tested two hypotheses: 1) that the effects of hypercholesterolemia on endothelial function in femoral arteries exceed those reported in brachial arteries and 2) that exercise (Ex) training enhances endothelium-dependent dilation and improves femoral artery blood flow (FABF) in hypercholesterolemic pigs. Adult male pigs were fed a normal fat (NF) or high-fat/cholesterol (HF) diet for 20 wk. Four weeks after the diet was initiated, pigs were Ex trained or remained sedentary (Sed) for 16 wk, thus yielding four groups: NF-Sed, NF-Ex, HF-Sed, and HF-Ex. Endothelium-dependent vasodilator responses were assessed in vivo by measuring changes in FABF after intra-arterial injections of ADP and bradykinin (BK). Endothelium-dependent and -independent relaxation was assessed in vitro by measuring relaxation responses to BK and sodium nitroprusside (SNP). FABF increased in response to ADP and BK in all groups. FABF responses to ADP and BK were not impaired by HF but were improved by Ex in HF pigs. BK- and SNP-induced relaxation of femoral artery rings was not altered by HF or Ex. To determine whether the mechanism(s) for vasorelaxation of femoral arteries was altered by HF or Ex, BK-induced relaxation was assessed in vitro in the absence or presence of N(G)-nitro-l-arginine methyl ester [l-NAME; to inhibit nitric oxide synthase (NOS)], indomethacin (Indo; to inhibit cyclooxygenase), or l-NAME + Indo. BK-induced relaxation was inhibited by l-NAME and l-NAME + Indo in all groups of femoral arteries. Ex increased the NOS-dependent component of endothelium-dependent relaxation in NF (not HF) arteries. Indo did not inhibit BK-induced relaxation. Collectively, these results indicate that hypercholesterolemia does not alter endothelial function in femoral arteries and that Ex training improves FABF responses to ADP and BK; however, the improvement cannot be attributed to enhanced endothelial function in HF femoral arteries. These data suggest that Ex-induced improvements in FABF in HF arteries are mediated by vascular adaptations in arteries/arterioles downstream from the femoral artery.     

Human coronary vascular smooth muscle and endothelium-dependent responses after storage at -75 degrees C.

In the present study, we investigated whether an established method of cryostorage at -75 degrees C in the presence of dimethyl sulfoxide (Me2SO) and fetal calf serum (FCS) could preserve the vascular and endothelial responses of isolated human coronary arteries. A total of 123 ring segments (4-5 mm in length) of epicardial coronary arteries were isolated within 1 to 2 h from hearts of four patients receiving a cardiac transplant. Thirty-nine coronary ring segments were studied immediately upon cleaning of surrounding tissues, while 84 similarly cleaned segments were stored at -75 degrees C for 7 to 10 days prior to in vitro reactivity studies. In the freshly isolated coronary arteries, addition of prostaglandin F2 alpha, endothelin (ET-1), or acetylcholine consistently produced a dose-dependent contraction, reaching a maximum contractile force of 9.6 +/- 0.7, 4.5 +/- 0.5, and 3.1 +/- 0.5 g (M +/- SEM), respectively, while histamine, thrombin and substance P consistently produced an endothelium-dependent relaxation (EDR) with a maximum of -89 +/- 2.8, -85 +/- 5.0, and -72 +/- 3.5%, respectively. Isoproterenol produced an endothelium-independent relaxation (-82 +/- 4.5%). Cryostorage of human coronary arteries at -75 degrees C without cryoprotectant resulted in a complete loss of the contractile response. In contrast, addition of Me2SO and FCS in the cryostorage medium significantly preserved the contractile responses, although they were decreased (1.9 +/- 0.3, 1.5 +/- 0.3, and 0.6 +/- 0.1 g to PGF2 alpha, ET-1, and acetylcholine, respectively) when compared to the fresh controls. The maximum EDR to histamine, thrombin, and substance P in the cryostored coronaries were also reduced to -40 +/- 5.6, -21 +/- 3.3, and -47 +/- 4.7%, respectively, and the isoproterenol-induced relaxation was reduced to -62 +/- 4.1%. These results suggest that although the cryostorage method described in the present report provided only limited preservation of human coronary arteries, significant vascular smooth muscle and endothelial-dependent functions were retained. Thus, it is possible that further refinement of the present cryostorage methodology may provide better preservation of functionally viable human blood vessels.     

Functional assessment of cryopreserved human aortas for pharmaceutical research

We evaluated the impact of standard cryopreservation on functional properties of human aortic homografts. From seven human donors, the thoracic descending aorta was obtained. Effects of cryopreservation on contractibility and endothelium function were tested. After cryopreservation no endothelium-dependent or endothelium-independent relaxation was found and the contractibility was strongly affected. Arteries showed no function and loss of endothelial integrity after cryopreservation and thawing.     

Different role of endothelium/nitric oxide in 17beta-estradiol- and progesterone-induced relaxation in rat arteries

The present study was aimed to examine the different role of endothelium/nitric oxide in relaxation induced by two female sex hormones, 17beta-estradiol and progesterone in rat isolated aortas and mesenteric arteries. The isometric force of each ring was measured with Grass force-displacement transducers in the organ bathes. 17beta-Estradiol induced both endothelium-dependent and -independent relaxation in the rat aortas but only the endothelium-independent relaxation in the rat mesenteric arteries. In contrast. progesterone induced both endothelium-dependent and -independent relaxation in the rat mesenteric arteries but only endothelium-independent relaxation in rat aortas. N(G)-Nitro-L-arginine methyl ester and methylene blue attenuated the relaxant response to 17beta-estradiol in the aortic rings or to progesterone in the mesenteric arteries. Pretreatment with L-arginine antagonized the effect of N(G)-nitro-L-arginine methyl ester on sex hormone-induced relaxation. The endothelium contribution to relaxation seems to only relate to lower concentrations of 17beta-estradiol and progesterone. In summary, the present results clearly demonstrate a different role of the functional endothelium in the relaxant response to 17beta-estradiol or progesterone in the conduit vessel (aorta) and the resistance vessels (mesenteric artery). Nitric oxide contributes largely to the endothelium-dependent relaxation induced by 17beta-estradiol in the isolated aortas or by progesterone in the mesenteric arteries.     

Chronic exercise training does not alter pulmonary vasorelaxation in normal pigs.

Exercise training increases acetylcholine-induced pulmonary vasorelaxation in pigs with coronary occlusion. The present study tested the hypothesis that chronic exercise training enhances endothelium-mediated vasorelaxation in pulmonary arteries from normal pigs. Yucatan miniswine exercised for 16 wk on a treadmill (Ex); control pigs (Sed) remained in pens. Pulmonary artery rings (2- to 3-mm OD) were studied using standard isometric techniques. Contractile responses to 80 mM KCl and norepinephrine (NE) were determined. Vessels were constricted with levels of NE that resulted in half-maximal contraction to examine endothelium-dependent relaxation to ACh and endothelium-independent relaxation to sodium nitroprusside in the presence and absence of nitric oxide synthase inhibition, cyclooxygenase inhibition, and endothelial denudation. Arteries from Ex pigs developed increased contraction to 80 mM KCl, but the response to NE did not differ between groups. Endothelium-dependent and endothelium-independent responses did not differ between Sed and Ex in the presence or absence of pharmacological inhibitors or denudation. We conclude that chronic exercise training does not alter endothelium-dependent or endothelium-independent vasorelaxation responses of pulmonary arteries from normal pigs.     

Chronic O2 exposure enhances vascular and airway smooth muscle contraction in the newborn but not adult rat.

Neonatal rats exposed to 60% O(2) for 14 days develop lung changes compatible with human bronchopulmonary dysplasia and pulmonary hypertension. Our aim was to evaluate and compare the newborn and adult rat pulmonary vascular and airway smooth muscle force generation and relaxation potential after exposure to 60% O(2) for 14 days. Vascular and airway intrapulmonary rings 100 microm in diameter were mounted on a myograph and bathed in Krebs-Henseleit solution bubbled with air- 6% CO(2) at 37 degrees C. Significant age-dependent changes in intrapulmonary arteries and their neighboring airway muscle properties were observed. Whereas hyperoxia enhanced force in neonatal vascular and airway muscle, the opposite was seen in adult samples. No changes in endothelium-dependent vascular relaxation were observed at either age, but the dose response to an endothelium-independent NO donor was altered. In the newborn experimental animals, the relaxation was reduced, whereas, in their adult counterparts, it was enhanced. After O(2) exposure, the bronchial muscle relaxation response to epithelium-dependent and -independent stimulation was not altered in either age group, whereas the epithelium-dependent response was decreased only in the adult. The antioxidant Trolox, or an endothelin-A and -B receptor antagonist, reversed the vascular and airway muscle's hyperoxia-induced changes. We conclude that chronic O(2) exposure in the newborn rat results in enhanced lung vascular and airway muscle contraction potential via a mechanism involving reactive oxygen species and the endothelin pathway. The present findings also suggest that the newborn is more susceptible to airway hyperresponsiveness after chronic O(2) exposure.     

Nitric oxide and prostacyclin-dependent pathways involvement on in vitro induced hypothermia

Nitric oxide and prostacyclin are endogenous endothelium-derived vasodilators, but little information is available on their release during hypothermia. This study was carried out to test the hypothesis that endothelium may modulate vascular reactivity to decreased temperature changes. Segments of contracted (prostaglandin F(2alpha), 2x10(-6)M) canine coronary, femoral, and renal arteries, with and without endothelium, were in vitro ("organ chambers") exposed to progressive hypothermia (from 37 to 10 degrees C) in graded steps. The study is limited to physiological measurements of vascular tone, in the presence or absence of PGI(2) and/or NOS inhibitors, which show correlation with the relaxation. Hypothermia induced vasodilatation of vessels with intact endothelium, which became endothelium-independent below 20 degrees C. This vasodilatation began at 35 degrees C and, in the presence of indomethacin (2x10(-6)M), at 30 degrees C. Endothelium-dependent vasodilatation to hypothermia was blocked by L-NMMA or L-NOARG (10(-5)M), two competitive inhibitors of nitric oxide synthase (n=5 each, P<0.05). Oxyhemoglobin (2x10(-6)M) also inhibited vasodilatation induced by hypothermia (n=6, P<0.05). Pretreatment with either atropine or pirenzepine (10(-6)M) inhibited hypothermia-mediated vasodilatation (n=5 each, P<0.05). The present in vitro study concluded that the endothelium is sensitive to temperature variations and indicated that PGI(2) and NO-dependent pathways may be involved endothelium-dependent relaxation to hypothermia. The endothelium-dependent vasodilatation to hypothermia, in systemic and coronary arteries, is mediated by the M1 muscarinic receptor.     

Chronic exercise training improves ACh-induced vasorelaxation in pulmonary arteries of pigs.

We hypothesized that exercise training would lead to enhanced endothelium-dependent vasodilation in porcine pulmonary arteries. Pulmonary artery rings (2- to 3-mm OD) were obtained from female Yucatan miniature swine with surgically induced coronary artery occlusion (ameroid occluder). Exercise training was performed for 16 wk, and vasomotor responses were studied by using standard isometric techniques. Contractile responses to 80 mM KCl, isosmotic KCl (10-100 mM), and norepinephrine (10(-8) to 10(-4) M) did not differ between sedentary (Sed) and exercise-trained (Ex) pigs. Relaxation was assessed to endothelium-dependent and endothelium-independent vasodilators after norepinephrine contraction. Pulmonary arteries of Ex pigs exhibited greater maximal relaxation to ACh (61.9 +/- 3.5%) than did those of Sed pigs (52.3 +/- 3.9%; P < 0.05). Endothelium-independent relaxation to sodium nitroprusside did not differ. Inhibition of nitric oxide synthase significantly decreased acetylcholine-induced relaxation, with greater inhibition in arteries from Ex pigs (P < 0.05). Inhibition of cyclooxygenase enhanced relaxation to acetylcholine in arteries from Sed pigs. We conclude that exercise training enhances endothelium-dependent (ACh-mediated) vasorelaxation in pulmonary arteries by mechanisms of increased reliance on nitric oxide and reduced production of a prostanoid constrictor.     

过氧亚硝基阴离子对离体兔肺动脉反应性变化的影响

探讨过氧亚硝基阴离子(peroxynitrite,ONOO^-)对离体兔肺动脉反应性变化的影响。用离体血管环技术观察ONOO^-孵育后肺动脉对钙离子载体A23187、ADP、ACh、硝普钠(sodium nitroprusside,SNP)和苯肾上腺素(phe-nylephrine,PE)的反应性张力变化。结果显示：(1)ONOO^-孵育后肺动脉对A23187、ADP和ACh引起的舒张反应明显降低,ONOO^-抑制内皮依赖受体依赖或受体非依赖性舒张反应有量效关系;(2)ONOO^-孵育可剂量依赖性抑制肺动脉对SNP的舒张反应;(3)0．5mmol／L ONOO^-孵育后肺动脉对PE的收缩反应明显增强,而1．0和2．0mmol／L ONOO^-导致肺动脉的收缩反应明显降低;(4)溶剂对肺动脉的反应性无明显影响,dec ONOO^-对PE和ADP的反应性影响不大,但可增强A23187、ACh和SNP的舒张反应。结果表明,ONOO^-可改变离体肺动脉的反应性。     

Short-term exercise training increases ACh-induced relaxation and eNOS protein in porcine pulmonary arteries.

We tested the hypothesis that short-term exercise (STEx) training and the associated increase in pulmonary blood flow during bouts of exercise cause enhanced endothelium-dependent vasorelaxation in porcine pulmonary arteries and increased expression of endothelial cell nitric oxide synthase (eNOS) and superoxide dismutase-1 (SOD-1) protein. Mature, female Yucatan miniature swine exercised 1 h twice daily on a motorized treadmill for 1 wk (STEx group, n = 7); control pigs (Sed, n = 6) were kept in pens. Pulmonary arteries were isolated from the left caudal lung lobe, and vasomotor responses were determined in vitro. Arterial tissue from the distal portion of this pulmonary artery was processed for immunoblot analysis. Maximal endothelium-dependent (ACh-stimulated) relaxation was greater in STEx (71 +/- 5%) than in Sed (44 +/- 6%) arteries (P < 0.05), and endothelium-independent (sodium nitroprusside-mediated) responses did not differ. Sensitivity to ACh was not altered by STEx training. Immunoblot analysis indicated a 3.9-fold increase in eNOS protein in pulmonary artery tissue from STEx pigs (P < 0.05) with no change in SOD-1 or glyceraldehyde-3-phosphate dehydrogenase protein levels. We conclude that STEx training enhances ACh-stimulated vasorelaxation in pulmonary arterial tissue and that this adaptation is associated with increased expression of eNOS protein.     

Endothelium-dependent and -independent relaxation in the forelimb and hindlimb vasculatures of swine

Limb differences in endothelial function exist between arm and leg vasculatures of humans. The current investigation tested the hypothesis that forelimb and hindlimb vasorelaxation are similar in the absence of limb differences in blood pressure. Conduit arteries (brachials/femorals) and second order arterioles were harvested from 22 miniature Yucatan swine. In vitro assessment of vasorelaxation was determined by administering increasing doses of bradykinin (BK), acetylcholine (ACh), and sodium nitroprusside (SNP). The role of the nitric oxide synthase (NOS) and cyclooxygenase (COX) pathways was assessed in conduit arteries but not resistance arterioles through L-NAME (300 microM) and INDO (5 microM) incubation, respectively. The relaxation responses to BK and ACh were similar in brachial and femoral arteries. SNP relaxation response was greater in the brachial compared to femoral arteries. There were also no significant differences in the relaxation responses of second order arterioles of the forelimb and hindlimb to BK, ACh, and SNP. Incubation of conduit arterial rings in L-NAME produced a greater reduction in BK and ACh relaxation in the brachial (approximately 25%) compared to femoral (approximately 13%) arterial rings. The current results of this investigation suggest that the forelimb and hindlimb vasculatures of swine have relatively similar vasorelaxation responses to both endothelium-dependent and -independent vasodilators.     

The Dipeptidyl Peptidase-4 Inhibitor Linagliptin Preserves Endothelial Function in Mesenteric Arteries from Type 1 Diabetic Rats without Decreasing Plasma Glucose

The aim of the study was to investigate the effect of the DPP-4 inhibitor linagliptin on the mechanism(s) of endothelium-dependent relaxation in mesenteric arteries from STZ-induced diabetic rats. Both normal and diabetic animals received linagliptin (2 mg/kg) daily by oral gavage for a period of 4 weeks. To measure superoxide generation in mesenteric arteries, lucigenin-enhanced chemiluminescence was used. ACh-induced relaxation of mesenteric arteries was assessed using organ bath techniques and Western blotting was used to investigate protein expression. Pharmacological tools (1μM TRAM-34, 1μM apamin, 100 nM Ibtx, 100 μM L-NNA, 10 μM ODQ) were used to distinguish between NO and EDH-mediated relaxation. Linagliptin did not affect plasma glucose, but did decrease vascular superoxide levels. Diabetes reduced responses to ACh but did not affect endothelium-independent responses to SNP. Linagliptin improved endothelial function indicated by a significant increase in responses to ACh. Diabetes impaired the contribution of both nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH) to endothelium-dependent relaxation and linagliptin treatment significantly enhanced the contribution of both relaxing factors. Western blotting demonstrated that diabetes also increased expression of Nox2 and decreased expression and dimerization of endothelial NO synthase, effects that were reversed by linagliptin. These findings demonstrate treatment of type 1 diabetic rats with linagliptin significantly reduced vascular superoxide levels and preserved both NO and EDH-mediated relaxation indicating that linagliptin can improve endothelial function in diabetes independently of any glucose lowering activity.     

Vasomotor responses of soleus feed arteries from sedentary and exercise-trained rats

Our goals were todetermine the nature of endothelium-dependent and -independent vascularresponses in isolated soleus feed arteries (SFA) and to test thehypothesis that these responses would be altered by exercise training.Exercise-trained rats ran 30 m/min, up a 15% grade, 1 h/day, 5 days/wkfor 10-12 wk, while sedentary control rats were confined to normalcage activity. SFA were isolated, cannulated, and pressurized at 90 cmH₂O. After a 1-h equilibrationperiod, the dose-response relationships to constrictors,endothelium-dependent dilators, and endothelium-independent dilatorswere examined. SFA developed spontaneous tone, demonstrated myogenicreactivity by maintaining vessel diameter in the face of large changesin intraluminal pressure, and constricted in a dose-dependent manner tonorepinephrine and potassium chloride. SFA dilated in a dose-dependentmanner to the endothelium-dependent dilators acetylcholine andincreased flow and to the endothelium-independent dilator sodiumnitroprusside. SFA did not dilate to the putative endothelium-dependentdilators bradykinin, substance P, and clonidine or to adenosine.Dilation to acetylcholine was attenuated markedly by arginine analogsand less by 20 mM KCl, but it was unaltered by indomethacin. Theseresults indicate that SFA respond to a number of vasoactive substances,consistent with the hypothesis that SFA participate in the control ofvascular resistance. However, exercise training does not appear toelicit a stimulus adequate to alter vasomotor responses in SFA.     

Acetylcholine causes endothelium-dependent contraction of mouse arteries

The goal of this study was to determine whether acetylcholine evokes endothelium-dependent contraction in mouse arteries and to define the mechanisms involved in regulating this response. Arterial rings isolated from wild-type (WT) and endothelial nitric oxide (NO) synthase knockout (eNOS(-/-)) mice were suspended for isometric tension recording. In abdominal aorta from WT mice contracted with phenylephrine, acetylcholine caused a relaxation that reversed at the concentration of 0.3-3 microM. After inhibition of NO synthase [with N(omega)-nitro-l-arginine methyl ester (l-NAME), 1 mM], acetylcholine (0.1-10 microM) caused contraction under basal conditions or during constriction to phenylephrine, which was abolished by endothelial denudation. This contraction was inhibited by the cyclooxygenase inhibitor indomethacin (1 muM) or by a thromboxane A(2) (TxA(2)) and/or prostaglandin H(2) receptor antagonist SQ-29548 (1 microM) and was associated with endothelium-dependent generation of the TxA(2) metabolite TxB(2.) Also, SQ-29548 (1 microM) abolished the reversal in relaxation evoked by 0.3-3 microM acetylcholine and subsequently enhanced the relaxation to the agonist. The magnitude of the endothelium-dependent contraction to acetylcholine (0.1-10 microM) was similar in aortas from WT mice treated in vitro with l-NAME and from eNOS(-/-) mice. In addition, we found that acetylcholine (10 microM) also caused endothelium-dependent contraction in carotid and femoral arteries of eNOS(-/-) mice. These results suggest that acetylcholine initiates two competing responses in mouse arteries: endothelium-dependent relaxation mediated predominantly by NO and endothelium-dependent contraction mediated most likely by TxA(2).     

Smooth muscle cell injury after cryopreservation of human thoracic aortas

The cryopreservation protocol we use for arterial reconstructive surgery has been studied to evaluate smooth muscle cell (SMC) structural integrity and viability before implantation. Samples of human thoracic aortas (HTA) were harvested from five multi-organ donors. Sampling included unfrozen and cryopreserved specimens. Cryopreservation was performed using RPMI with human albumin and 10% Me(2)SO in a controlled-rate freezing apparatus. Thawing was accomplished by submerging bags in a water bath (39 degrees C) followed by washings in cooled saline. In situ cell preservation as investigated by light and transmission electron microscopy showed that SMCs from cryopreserved HTA had nuclear and cytoplasmic changes. A TUNEL assay, performed to detect DNA fragmentation in situ, showed increased SMC nuclear positivity in cryopreserved HTA when compared to unfrozen samples. 7-AAD flow cytometry assay of cells derived from cryopreserved HTA showed that an average of 49+/-16% cells were unlabeled after cryopreservation. Organ cultures aimed to study cell ability to recover cryopreservation damage showed a decreasing number of SMCs from day 4 to day 15 in cryopreserved HTA. In conclusion, the cryopreservation protocol applied in this study induces irreversible damage of a significant fraction of arterial SMCs.     

Effect of tumor necrosis factor on hypoxic pulmonary vasoconstriction.

The effects of tumor necrosis factor (TNF) on hypoxic pulmonary vasoconstriction (HPV) and endothelium-dependent relaxation were examined in a blood-perfused rat lung preparation. Lungs from TNF-treated rats (0.26 mg/kg iv 12 h before experimentation) had a significantly greater HPV and a reduced vasorelaxant response to the endothelium-dependent vasodilator acetylcholine (ACh) but a similar vasorelaxant response to the endothelium-independent vasodilator nitroprusside compared with lungs from control rats (pretreated with 0.1 ml saline iv). Pentoxifylline (20 mg/kg iv and ip 20 min before administration of TNF) had no detectable effect on either HPV or ACh-induced relaxation but completely negated the augmentation on HPV and the inhibiting action on ACh-induced relaxation caused by TNF. The TNF effect on ACh relaxation was unaffected by pretreatment with L-arginine. These results indicate that TNF induces endothelial dysfunction and enhances HPV, effects that are inhibited by pentoxifylline.