Morphology and function of dog arterial grafts preserved in UW-solution

Objectives:To assess the function of arterial grafts after prolonged preservation in the University of Wisconsin solution (UW), in vitro and in vivo.Methods:Carotid arteries were harvested from dogs and stored for 1–21 days at 4°C in UW (n = 10) or in PBS (0.9% NaCl, pH 7.4), (PBS) (n = 10). Slices were examined by lightmicroscopy (LM) and scanning electron microscopy (SEM). For viability testing, specimens were connected to an isometric force transducer (2 × n = 9). Contractile and relaxation responses were examined by adding phenylephrine (200μM) and metacholine (200μM), respectively. For in vivo studies (n = 41), 2.5cm carotid artery segments were implanted orthotopically, as autografts and allografts, after 14 days of storage in UW or in PBS. Autologous veins were used as controls. After 28 days or 56 days, arteriography was performed and the grafts were excised for LM and SEM.Results:The arterial endothelial layer remained intact after up to 14 days of storage in UW. In PBS, the endothelium was lost after 3 days. The functional response after 14 days storage in UW was approximately 50% vs. 0% after 14 days in PBS. In the autografts, total patencies (28 days + 56 days) were 100% (8/8) and 63% (5/8) for UW and PBS stored grafts, respectively. In the allografts, the UW and PBS preserved grafts showed total patencies of 86% (12/14) and 83% (5/6), respectively. Microscopically, the allografts showed fibrotic degeneration.Conclusions:Arteries are well preserved in UW up to 14 days of storage. Arterial autografts preserved in UW showed good patency and better integrity of the vessel wall after implantation, than grafts stored in PBS or allografts (without immunosuppressive therapy)

The shared frameshift mutation landscape of microsatellite-unstable cancers suggests immunoediting during tumor evolution

The immune system can recognize and attack cancer cells, especially those with a high load of mutation-induced neoantigens. Such neoantigens are abundant in DNA mismatch repair (MMR)-deficient, microsatellite-unstable (MSI) cancers. MMR deficiency leads to insertion/deletion (indel) mutations at coding microsatellites (cMS) and to neoantigen-inducing translational frameshifts. Here, we develop a tool to quantify frameshift mutations in MSI colorectal and endometrial cancer. Our results show that frameshift mutation frequency is negatively correlated to the predicted immunogenicity of the resulting peptides, suggesting counterselection of cell clones with highly immunogenic frameshift peptides. This correlation is absent in tumors with Beta-2-microglobulin mutations, and HLA-A*02:01 status is related to cMS mutation patterns. Importantly, certain outlier mutations are common in MSI cancers despite being related to frameshift peptides with functionally confirmed immunogenicity, suggesting a possible driver role during MSI tumor evolution. Neoantigens resulting from shared mutations represent promising vaccine candidates for prevention of MSI cancers. DNA mismatch repair (MMR)-deficient cancers with microsatellite-instability are characterized by a high load of frameshift mutation-derived neoantigens. Here, by mapping the frameshift mutation landscape and predicting the immunogenicity of the resulting peptides, the authors show evidence of immunoediting in MMR-deficient colorectal and endometrial cancers.Peer reviewe

Vasopressin-induced presynaptic facilitation of sympathetic neurotransmission in the pithed rat

Objective Several studies have shown that arginine vasopressin (AVP) potentiates the sympathetic nervous transmission in isolated vessels. The present study investigates such a potentiation in the pithed rat model. Methods Male Wistar rats weighing 270-310 g were used. Spinal-cord stimulation was applied, with frequencies of 0.25-4 Hz, in the presence or absence of a subpressor dose of intravenous (i.v.) AVP (1 pmol/kg per min). In addition, the effect of AVP on postsynaptic alpha-adrenoceptor-mediated responses was studied using exogenously administered noradrenaline (NA). For this purpose dose-response curves (DRCs) for NA (i.v.) were constructed. Results In the pithed rat model endogenously generated angiotensin II facilitates neurally mediated increments in vascular resistance. Without the administration of the angiotensin II type 1 (AT(1)) antagonist irbesartan, the facilitating effect of AVP was not visible. However, after the administration of the AT(1) antagonist irbesartan, the facilitating effect of AVP became apparent. The stimulation-induced rise in diastolic blood pressure (DBP) was enhanced in the presence of AVP from 63.7 +/- 4.5 to 78.6 +/- 4.2 mmHg, at a stimulation frequency of 4 Hz. The vasopressin receptor V-1 antagonist, SR-49059, completely inhibited this AVP-inDuced facilitation, whereas the V-2 antagonist, SR-1211463B, or the V-2 agonist desmopressin, did not. The DRC of exogenously administered NA was not influenced by AVP. Conclusion The stimulating effect of AVP on sympathetic neurotransmission is completely dependent on the stimulation of presynaptically located V-2 receptors. The facilitating effect of angiotensin II on the sympathetic nervous system (SNS) in the pithed rat model masks the facilitating effect of AVP in this preparation. (C) 2002 Lippincott Williams Wilkin

Sympatho-inhibitory actions of irbesartan in pithed spontaneously hypertensive and Wistar-Kyoto rats

Angiotensin If (Ang 11) can enhance sympathetic neurotransmission by acting on (AT,) receptors that are located on sympathetic nerve terminals. We investigated presynaptic blockade by the selective AT(1)-receptor antagonist irbesartan in pithed spontaneously hypertensive rats and normotensive Wistar-Kyoto rats (WKY). We compared the presynaptic inhibitory dose with that required for the blockade of AT(1)-receptors on vascular smooth muscle in both strains. To investigate blockade of presynaptic AT(1)-receptors, we studied the effect of irbesartan on the sequelae of electric stimulation of the thoraco-lumbar sympathetic outflow (0.25-8 Hz). To study the interaction between postsynaptic AT(1)-blockers and alpha-adrenoceptors, the effects of irbesartan on pressor responses to exogenous noradrenaline (NA) were established. Additionally, we studied the effect of irbesartan on dose-response curves for the vasocontriction induced by exogenous Ang II. Pressor responses to electrical stimulation of thoracolumbar sympathetic neurones, to exogenous Ang 11, as well as to (NA) were enhanced in spontaneously hypertensive rats (SHR) compared with WKY. The stimulation-induced rise in DBP could be dose-dependently reduced by irbesartan (0.3-10 mg/kg) in both SHR and WKY. the pIC50 values (doses which suppress the rise in DBP by 50% compared with control) were 5.60 +/- 0.09 and 5.72 +/- 0.08 for SHR and WKY, respectively (P > 0.05). In SHR, no effect of irbesartan (3 mg/kg) on pressor responses to exogenous NA was observed. In contrast, in WKY, irbesartan (3 mg/kg) caused a rightward shift of the dose-response curve to exogenous NA. Irbesartan (0.3-3 mg/kg) caused a depression of E-max values and a rightward shift of the dose-response curves to exogenous Ang 11 in a similar fashion in both SHR and WKY. From these results we conclude thatboth in SHR and in WKY, Ang 11 exerts a facilitatory effect on sympathetic neurotransmission, which is mediated by prejunctional AT(1)-receptors in both strains. Irbesartan displays comparable sympatho-inhibitory potency in the normotensive and hypertensive pithed rat preparations. A facilitatory effect via postsynaptically located AT(1)-receptors on alpha-adrenoceptor-mediated responses exists in WKY, but not in SHR. In both strains the required dose to inhibit presynaptic effects is somewhat higher than the dose required to inhibit postsynaptic effects. No differences, therefore, seem to exist between the two strains regarding the affinity of irbesartan for pre- and postjunctional AT(1)-receptors, respectivel

The influence of endogenously generated reactive oxygen species on the inotropic and chronotropic effects of adrenoceptor and ET-receptor stimulation

Reactive oxygen species (ROS) play a role in cardiovascular diseases such as heart failure and hypertension. Furthermore, increasing evidence has accumulated suggesting that ROS can also be formed subsequent to the stimulation of various receptors, thus functioning as second messengers. The objective of the present study was to elucidate the role of intracellular-generated ROS in the inotropic and chronotropic effects of the alpha(1)- and beta-adrenoceptor and the ET-receptor stimulation in isolated rat atria. In addition, we investigated whether the MAPK(erk) pathway is involved in the ROS-provoked rise of contractile force. For this purpose hydrogen peroxide was applied, which is known to serve several endogenous functions as a second messenger. Moreover, hydrogen peroxide readily crosses cell membranes, which thus allows to mimic the intracellular formation. Preincubation of atria with EUK 8 (400 muM), a cell permeable superoxide dismutase- and catalase-mimetic, reduced the positive inotropic effect upon alpha(1)-adrenoceptor and ET-receptor stimulation. The responsiveness to beta-adrenoceptor stimulation remained unaffected by this pretreatment. The chronotropic effects were not altered by preincubation with EUK 8. In contrast to the MAPK(p38) inhibitor SB203580 (2 and 10 muM), the two MKKmek inhibitors PD98059 (30 and 100 muM) and U0126 (10 muM) significantly attenuated the positive inotropic response to hydrogen peroxide in isolated rat left atria. In addition, inhibition of the Na+/H+ exchange (NHE) by cariporide (1 muM) counteracted ROS-provoked increase of contractile force. From the present study we conclude that the inotropic responses to alpha(1)-adrenoceptor and ET-receptor stimulation are, at least partially, caused by intracellular-formed ROS, that subsequently may activate the MAPK(erk) pathway and the NH

Effects of hypochlorite and hydrogen peroxide on cardiac autonomic receptors and vascular endothelial function

1. Reactive oxygen species (ROS) are known to be involved in the progression of various cardiovascular diseases. One source of ROS is activated neutrophils, which can release superoxide anion radicals and hydrogen peroxide by membrane-bound NAD(P)H oxidases. These ROS not only destroy bacteria, but may also affect mammalian tissue. In addition, hydrogen peroxide serves as a substrate for myeloperoxidase, an enzyme that is released by activated neutrophils during inflammatory processes, as seen, for instance, in reperfusion injury and atherosclerosis. Myeloperoxidase catalyses the oxidation of chloride by hydrogen peroxide, yielding hypochlorite, an extremely potent oxidant. 2. The purpose of the present study was to evaluate the effects of hypochlorite on a variety of receptor-dependent processes in rat isolated left atria and rat thoracic aorta and to compare these results with the phenomena observed after incubation with hydrogen peroxide. 3. In the presence of hypochlorite (300 mumol/L), the positive inotropic response of alpha(1) -adrenoceptor stimulation by methoxamine (300 mumol/L) was converted into a negative inotropic response. In contrast, the positive inotropic effects of the beta(1) /beta(2) -adrenoceptor agonist isoprenaline (3 mumol/L) and endothelin (ET)-1 (100 nmol/L) remained largely unaffected. 4. The inversion of alpha(1) -adrenoceptor-mediated inotropy was not obtained in the presence of hydrogen peroxide (500 mumol/L). Hydrogen peroxide did not affect the positive inotropic response of isoprenaline, but it completely abolished the inotropic effect of ET-1. 5. The effect of cardiac M-2 -receptor stimulation was studied in the presence of hypochlorite and hydrogen peroxide. The negative inotropic response to acetylcholine (ACh) was significantly enhanced after hypochlorite incubation compared with control. 6. In the rat thoracic aorta, endothelial function, evaluated by means of ACh-induced vasodilation, was completely abolished in the presence of hypochlorite (100 mumol/L), but remained unaffected by treatment with the same concentration of hydrogen peroxide. 7. From these data, we conclude that hypochlorite exerts more toxic properties than its precursor hydrogen peroxide, leading to substantial physiological alterations in cardiac and vascular tissu

Influence of the nature of pre-contraction on the responses to commonly employed vasodilator agents in rat-isolated aortic rings

The relaxing properties of vasodilator drugs in vitro may depend on the characteristics of the contractile state of the vessel investigated. Rat-isolated thoracic aortas were exposed to different types of pre-contraction. The following vasoconstrictor agents were used: phenylephrine (PhE), a selective alpha(1)-adrenoceptor agonist; St 587, a partial alpha(1)-adrenoceptor stimulant; U46619 (U-46), a thromboxane A(2) agonist; and potassium ions causing receptor-independent depolarization of the membrane. After pre-contraction, various differential vasodilator drugs were investigated: methacholine (MCh, endothelium dependent), sodium nitroprusside (SNP, NO donor), forskolin (FSK, adenylyl cyclase stimulant) and nifedipine, a Ca2+-antagonist (selective L-type calcium antagonist). The vasodilator activity of these compounds was quantified by their vasodilator potency value (pD(2)) and efficacy (E-max) obtained from their concentration-response curves. PhE (0.1, 0.3, 3 mu(M)) caused isometric responses of 4.8 +/- 0.3, 6.5 +/- 0.3 and 7.8 +/- 0.5 mN, respectively. An increase of the PhE concentration from 0.1 to 3 mu(M) did not influence the response to FSK while it reduced the pD(2) of SNP (8.6 +/- 0.1 to 7.35 +/- 0.1). Under these conditions, only the E-max of MCh was reduced (96.3 +/- 4.3% to 43.3 +/- 6.9%). U46 (0.18, 0.3, 1 mu(M)) increased the contractile force by 7.4 +/- 0.4, 8.8 +/- 0.3 and 10.4 +/- 0.3 mN, respectively. Increasing the concentration of U-46 from 0.18 to 1 mu(M) affected only the efficacy of SNP (84 +/- 4.4% to 17 +/- 8.8%) and MCh (64.5 +/- 12.3% to 0.0 +/- 9.2%) and reduced the potency of FSK (7.9 +/- 0.26 to 7.15 +/- 0.10). The concentration of K+-ions from 25 to 30 and 40 mm increased the contractile force by 4.0 +/- 0.4, 7.0 +/- 0.5 and 10.8 +/- 0.4 mN, respectively. The increase in [K+] caused a potency decrease of FSK (7.1 +/- 0.0 to 5.8 +/- 0.0) whereas both efficacy and potency were reduced for SNP (95.6 +/- 1.8% to 65.8 +/- 1.9% and 8.7 +/- 0.1 to 7.2 +/- 0.1) and MCh (55.4 +/- 3.5% to 24.5 +/- 0.8% and 7.4 +/- 0.3 to 6.1 +/- 0.4). Inhibiting of the endothelial NO production by L-NAME 100 mu(M) resulted after pre-contraction with PhE and potassium in comparable differences in properties for SNP. Pre-contraction with St 587 1, 3, 10 and 30 mu(M) shows comparable results after nifedepine relaxation. The present experiments clearly demonstrate that the characteristics of the applied pre-contraction strongly, but differentially influence both the potency and efficacy of various vasodilator drugs in vitro. Accordingly, in vitro characterization of vasodilator drugs should be performed under a carefully standardized protocol of pre-contractio