Birgit Zatschler

Improved vessel preservation after 4 days of cold storage: Experimental study in rat arteries

BACKGROUND Cold storage of arteries for reconstructive and bypass surgery may result in injury of endothelial cells which may promote low perfusion and graft vasculopathy. METHODS A recently developed N-acetyl histidine-buffered, potassium-chloride enriched, and amino acid-fortified vascular storage solution augmented with iron chelators deferoxamine (100 micromol/L) and LK 614 (20 micromol/L) was studied in the rat superior mesenteric artery and aorta with respect to: (1) potassium-induced vessel tone, (2) endothelium-dependent and -independent relaxation, and (3) endothelial nitric oxide synthase (eNOS) protein expression over 4-days cold storage (4 degrees C).This solution was compared with traditional storage solutions, histidine-tryptophan-ketoglutarate (HTK) and physiological saline solution (PSS). RESULTS Vessels stored for 4 days in the new solution were significantly better protected than those stored in traditional HTK or PSS. The protective effects comprised: (1) vessel tone development after stimulation with potassium-chloride solution, (2) endothelium-dependent and -independent vessel relaxation, and (3) eNOS expression. With iron chelators (deferoxamine 100 microM, LK 614 20 microM) present in the storage solution, endothelium-dependent relaxations (eNOS-dependent and K(Ca)-channel-dependent) were fully maintained after 96 hours of cold storage. Endothelial cell structure was significantly better maintained after 96 hours in the new solution than in HTK or PSS solutions. Already, 2 hours of cold storage in HTK resulted in a significant loss of structurally intact endothelium. The structural changes correlated significantly with the diminished vessel relaxation capacity. Furthermore, tissue reductive capacity was only preserved after 96 hours storage if the new solution was used. CONCLUSION The new storage solution is superior to traditional HTK and PSS cold storage with respect to: (1) preservation of vessel structure and function; (2) the presence of iron chelators significantly improved protection of endothelial function; and (3) the new solution permits cold vessel storage for a minimum of 4 days with full maintenance of endothelial function and its coupling to smooth muscle.

Even short-time storage in physiological saline solution impairs endothelial vascular function of saphenous vein grafts

OBJECTIVES A faultless endothelial layer is decisive for vascular function and therewith grafts patency. Functional impairment of the endothelium increases risk of graft thrombosis, intimal hyperplasia, and consecutive accelerated graft atherosclerosis. Storage solutions for intra-operatively harvested saphenous vein segments (SVS) might have significant impact on endothelial function. We investigated the impact of short-time storage in physiological saline solution (PSS) and a potassium-chloride- and N-acetylhistidine-enriched storage solution on venous endothelial function. METHODS Intra-operatively isolated SVSs (n=19) were stored in different storage solutions for 90 min. They were then immediately studied in tissue bath at 36°C with continuous oxygen insufflation. Following preconstriction with norepinephrine, dose-response relaxation curves of bradykinine (Brad) and sodium nitroprusside (SNP) were determined. We compared developed maximum wall tension, vessel constriction kinetics, endothelial cell- and smooth muscle cell (SMC)-dependent vasodilatory function. RESULTS Maximum vessel wall tension was reduced significantly in PSS-stored vessels (10.1 ± 9.8 mN mm(-1) vs 3.5 ± 3.4 mN mm(-1); p=0.0372). Endothelium-derived vasodilatory function was likewise significantly reduced after short-time storage (20.6 ± 34.4% vs 35.0 ± 27.0%; p=0.0437). SNP-mediated SMC-vasodilatory function was maintained equally well in both groups (88.2 ± 21.8% vs 83.0 ± 30.6% in PSS; p=n.s.). CONCLUSION Even short-time storage in PSS significantly impairs endothelial vascular function. Concerning the essential role of a faultless endothelial layer, the quite common use of PSS as a storage solution for SVSs in CABG surgery has to be discussed critically.

Preservation of human artery function following prolonged cold storage with a new solution

OBJECTIVE Blood vessels are an important tissue for allogenic vessel replacement surgery, which is needed for example following infection of artificial grafts. For tissue banking, European legislation requires evidence of tissue sterility with assays performed over 1 week. Currently, used cold storage solutions do not protect vascular function longer than 2 days. This does not allow completion of microbiological testing. This discrepancy has almost completely stopped vessel banking in Europe. METHODS We compared the recently developed storage solution TiProtec (Dr F Köhler Chemie, Bensheim, Germany) with traditionally used histidine-tryptophan-ketoglutarate (HTK) solution, 0.9% NaCl, and phosphate-buffered saline (physiological saline solution [PSS]) solution for extended cold (4°C) storage up to 25 days. Isolated rings of human internal mammary artery were studied with respect to several parameters of vessel function, including vessel tone development, endothelium-dependent and endothelium-independent relaxation, and tissue reductive capacity. RESULTS Vessels stored in NaCl or PSS for ≥10 hours failed to develop tone after rewarming. Mammary arteries stored in HTK for 4 hours at 4°C initially showed a well-preserved vessel function with respect to vessel tone development, as well as endothelial and smooth muscle dilatative function. However, following 4 days of cold storage, vessel tone development and dilatative responses were significantly impaired. In contrast, arteries stored in TiProtec showed full preservation of vessel tone as well as endothelial and smooth muscle function after 4 days of cold storage. Even after 10 days of cold storage, endothelium-dependent relaxation was approximately 50% of control, and smooth muscle function was fully preserved. Over 2 weeks, tissue reductive capacity was significantly better maintained after cold storage in TiProtec compared with vessels stored in NaCl. CONCLUSIONS In contrast to traditional HTK, NaCl, or PSS storage, TiProtec solution offers an excellent potential for prolonged cold storage of human arteries, which may close the existing gap between legal requirements for tissue banking and current cold preservation methods.

Preservation of endothelial vascular function of saphenous vein grafts after long-time storage with a recently developed potassium-chloride and N-acetylhistidine enriched storage solution.

OBJECTIVES Saphenous vein grafts are still commonly used in cardiac, vessel and also in transplant surgery. In cardiac surgery, a desperate graft situation could force to keep nonutilized explanted vein segments after CABG in reserve for case of early graft failure. Historically there were no options for adequate long-time graft storage protecting the endothelial layer with its important antithrombotic and immunosuppressive functional aspects. Commonly isotonic saline solution (sodium chloride [NaCl]) has been used as a storing solution in this case. We investigated the impact of long-time storage in NaCl and a recently developed potassium-chloride and N-acetylhistidine enriched storage solution (TiProtec, Dr. Köhler Chemie, Germany) on endothelial function of saphenous veins. METHODS Saphenous vein segments (n = 19) were intraoperatively isolated and stored for 24 and 96 hours. The segments were examined in a Mulvany-myograph to assess vessel function. Following preconstriction with norepinephrine, dose-response curves were assessed for relaxation with bradykinin and sodium-nitroprusside. We compared developed maximum wall tension and endothelial cell and smooth muscle cell (SMC) dependent vasodilatory function. RESULTS Maximum vessel wall tension was significantly better preserved in TiProtec-stored vessels after 24 h in comparison to segments stored in NaCl (5.11 ± 4.79 mN/mm vs. 2.48 ± 2.43 mN/mm; p = 0.033) and 96 h (4.94 ± 2.82 mN/mm vs. 2.80 ± 1.76 mN/mm; p = 0.042). Likewise endothelium-derived vasodilatory function was maintained significantly after 24 hours in TiProtec-stored vessels (36.9 ± 2.6% vs. 11.8 ± 30.9%; p = 0.005). After 96 hours, endothelium-dependent vascular function was nearly abolished in NaCl-stored vessels, but largely preserved in TiProtec-stored segments (20.6 ± 2.9% vs. 1.9 ± 4.3% in NaCl; p = 0.015). Sodium nitroprusside-mediated SMC-vasodilatory function was better maintained after 24 hours of storage in TiProtec group (88.8 ± 6.4% vs. 61.3 ± 8.2%; p = 0.009). After 96 hours of storage, SMC relaxation did not significantly differ between both storage groups which might be due to a distinct reduction of contractile function in NaCl-stored vessels (98.6 ± 5.0% and 77.9 ± 10.5% for Tiprotec and NaCl, respectively). CONCLUSION Vessel functions comprising contraction, endothelium-dependent and -independent vasodilatation are significantly reduced following 24 hours of cold storage in NaCl. After 96 hours of storage in NaCl these functions are nearly totally abolished. TiProtec is able to largely reduce this loss of function during cold storage. Therefore, TiProtec is a feasible option for longer term storage of saphenous vein grafts in CABG vessel and transplant surgery.

A novel role of endothelium in activation of latent pro-membrane type 1 MMP and pro-MMP-2 in rat aorta

AIMS Aortic stiffness is an independent risk factor for progression of cardiovascular diseases. Degradation of elastic fibres in aorta due to angiotensin II (ANGII)-stimulated overactivation of latent membrane type 1 matrix metalloproteinase (MT1MMP) and matrix metalloproteinase-2 (MMP2) is regarded to represent an important cause of aortic stiffness. Therefore, clarification of the causal mechanisms triggering the overactivation of these MMPs is of utmost importance. This study addresses the endothelium as a novel key activator of latent pro-MT1MMP and pro-MMP2 in rat aorta. METHODS AND RESULTS Using a co-culture model of rat aortic endothelial cells (ECs) and smooth muscle cells (SMCs), we found that ANGII stimulation resulted in activation of latent pro-MT1MMP and pro-MMP2 in SMCs exclusively when co-cultured with ECs (assessed with western blot and gelatin zymography, respectively). EC-specific AT1 receptor stimulation triggered endothelin-1 release and paracrine action on SMCs. Endothelin-1 increased expression and activity of pro-protein convertase furin in SMCs via endothelin receptor type A (assessed with qPCR and furin activity assay, respectively). Consequently, furin acted in two ways. First, it increased the activation of latent pro-MT1MMP and, second, it activated pro-αvβ3 integrin. Both pathways led to overactivation of latent pro-MMP2. In vitro findings in the co-culture model were fully consistent with the ex vivo findings obtained in isolated rat aorta. CONCLUSIONS We propose that the endothelium under ANGII stimulation acts as a novel and key activator of latent pro-MT1MMP and pro-MMP2 in SMCs of rat aorta. Therefore, endothelium may critically contribute to pathophysiology of aortic stiffness.

Heparinized blood better preserves cellular energy charge and vascular functions of intraoperatively stored saphenous vein grafts in comparison to isotonic sodium-chloride-solution

OBJECTIVE Several studies have addressed the optimal storage conditions for vascular grafts during surgery. The results remain contradictionary. This may be attributed to the fact, that the various vascular beds have a different sensitivity to storage. We analyzed the impact of storage in isotonic saline solution (NaCl) or heparinized blood the vascular functions of human saphenous vein grafts. Special care was taken to choose storage conditions which are relevant for intraoperative storage of a saphenous vein graft in a setting of coronary artery bypass grafting with vein and internal mammary artery as grafts. METHODS Intraoperatively isolated V. saphena-segments (n = 36) were stored in NaCl or heparinized blood for approximately 30 minutes at room temperature. Subsequently, the segments were examined in a Mulvany-myograph. Following preconstriction with norepinephrine, concentration-relaxation curves were assessed for bradykinin and sodium-nitroprusside to assess developed vessel-wall tension as well as endothelium- and smooth-muscle-cell dependent vasorelaxation. The availability of adenosintriphosphate (energy charge) was determined based on liquid chromatography measurements of nucleotide tissue levels. RESULTS Mean storage time was 27.4 ± 2.4 min in NaCl- and 26.3 ± 2.7 min in blood-group, respectively. After this period, receptor-dependent and-independent maximum of developed vessel wall tension was significantly reduced in NaCl-group (p = 0.05 and p = 0.045, respectively). Furthermore, the energy charge was significantly (p = 0.046) better preserved after blood storage (74 ± 1%) in comparison to NaCl-group (68 ± 2%). Endothelium-induced vasodilatation in response to bradykinin reached only 12.3 ± 2.5% in NaCl-group, but 19.3 ± 5.2% in blood-group (p = 0.033). Alike, EC50-concentration of bradykinin for half-maximal relaxation was significantly lower in blood- than in NaCl-group (log EC50 -7.08 ± 0.3 and -5.91 ± 0.4; respectively; p = 0.046). Endothelium-independent smooth muscle relaxation in response to sodium-nitroprusside was not different between both groups. CONCLUSION Heparinized blood better preserves vascular contractile and endothelial functions of the saphenous vein graft. Storage in NaCl rapidly compromises vascular functions and impaires cellular energy. NaCl should no longer be recommended for intraoperative storage of harvested V. saphena grafts.

A differential impact of lithium on endothelium-dependent but not on endothelium-independent vessel relaxation.

Lithium is drug for bipolar disorders with a narrow therapeutic window. Lithium was recently reported to prevent stroke and protect vascular endothelium but tends to accumulate particularly in the brain and kidney. Here, adverse effects are common; however mechanisms are still vaguely understood. If lithium could also negatively influence the endothelium is unclear. We hypothesize that at higher lithium levels, the effects on endothelium reverses--that lithium also impairs endothelial-dependent relaxation of blood vessels. Vessel grafts from de-nerved murine aortas and porcine middle cerebral arteries were preconditioned using media supplemented with lithium chloride or acetate (0.4-100 mmol/L). Native or following phenylephrine-induced vasoconstriction, the relaxation capacity of preconditioned vessels was assessed by isometric myography, using acetylcholine to test the endothelium-dependent or sodium nitroprusside to test the endothelium-independent vasorelaxation, respectively. At the 0.4 mmol/L lithium concentration, acetylcholine-induced endothelium-dependent vessel relaxation was slightly increased, however, diminished in a concentration-dependent manner in vessel grafts preconditioned with lithium at higher therapeutic and supratherapeutic concentrations (0.8-100 mmol/L). In contrast, endothelium-independent vasorelaxation remained unaltered in preconditioned vessel grafts at any lithium concentration tested. Lithium elicits opposing effects on endothelial functions representing a differential impact on the endothelium within the narrow therapeutic window. Lithium accumulation or overdose reduces endothelium-dependent but not endothelium-independent vasorelaxation. The differentially modified endothelium-dependent vascular response represents an additional mechanism contributing to therapeutic or adverse effects of lithium.

Whey peptide Isoleucine-Tryptophan inhibits expression and activity of matrix metalloproteinase-2 in rat aorta

Aortic stiffness is an independent risk factor for development of cardiovascular diseases. Activation of renin-angiotensin-aldosterone system (RAAS) including angiotensin converting enzyme (ACE) activity leads to overproduction of angiotensin II (ANGII) from its precursor angiotensin I (ANGI). ANGII leads to overexpression and activation of matrix metalloproteinase-2 (MMP2), which is critically associated with pathophysiology of aortic stiffness. We previously reported that the whey peptide Isoleucine-Tryptophan (IW) acts as a potent ACE inhibitor. Herein, we critically elucidate the mechanism of action by which IW causes inhibition of expression and activity of MMP2 in aortic tissue. Effects of IW on expression and activity of MMP2 were assessed on endothelial and smooth muscle cells (ECs and SMCs) in vitro and ex vivo (isolated rat aorta). As controls we used the pharmaceutical ACE inhibitor - captopril and the ANGII type 1 receptor blocker - losartan. In vitro, both ANGII and ANGI stimulation significantly (P<0.01) increased expression of MMP2 assessed with western blot. Similarly, to captopril IW significantly (P<0.05) inhibited ANGI, but not ANGII mediated increase in expression of MMP2, while losartan also blocked effects of ANGII. Signaling pathways regulating MMP2 expression in ECs and SMCs were similarly inhibited after treatment with IW or captopril. In ECs IW significantly (P<0.05) inhibited JNK pathway, whereas in SMCs JAK2/STAT3 pathway, assessed with western blot. In vitro findings were fully consistent with results in isolated rat aorta ex vivo. Moreover, IW not only inhibited the MMP2 expression, but also its activation assessed with gelatin zymography. Our findings demonstrate that IW effectively inhibits expression and activation of MMP2 in rat aorta by decreasing local conversion of ANGI to ANGII. Thus, similar to pharmaceutical ACE inhibitor captopril the dipeptide IW may effectively inhibit ACE activity and prevent the age and hypertension associated rise of aortic stiffness.

Effects of tryptophan-containing peptides on angiotensin-converting enzyme activity and vessel tone ex vivo and in vivo

PurposeOver-activation of the renin-angiotensin axis and worsening of vascular function are critical contributors to the development of hypertension. Therefore, inhibition of angiotensin-converting enzyme (ACE), a key factor of the renin-angiotensin axis, is a first line treatment of hypertension. Besides pharmaceutical ACE inhibitors, some natural peptides have been shown to exert ACE-inhibiting properties with antihypertensive effects and potentially beneficial effects on vascular function. In this study, the ACE-inhibiting potential and effects on vascular function of tryptophan-containing peptides were evaluated.MethodsThe ACE inhibitory action and stability of tryptophan-containing peptides was tested in endothelial cells—a major source of whole body ACE activity. Furthermore, the efficacy of peptides on vascular ACE activity, as well as vessel tone was assessed both ex vivo and in vivo.ResultsIn human umbilical vein endothelial cells (HUVEC), isoleucine-tryptophan (IW) had the highest ACE inhibitory efficacy, followed by glutamic acid-tryptophan (EW) and tryptophan-leucine (WL). Whereas none of the peptides affected basal vessel tone (rat aorta), angiotensin I-induced vasoconstriction was blocked. IW effectively inhibited aortic ACE activity ex vivo taken from SHRs after 14-weeks of oral treatment with IW. Furthermore, IW treated SHRs showed better endothelium-dependent vessel relaxation compared to placebo.ConclusionThis study shows strong ACE-inhibiting effects of IW, EW and WL in HUVECs and aorta. The peptides effectively counteract angiotensin-induced vasoconstriction and preserve endothelium-dependent vessel relaxation. Thus, tryptophan-containing peptides and particularly IW may serve as innovative food additives with the goal of protection from angiotensin II-induced worsening of vascular function.

Sex-specific differences in age-dependent progression of aortic dysfunction and related cardiac remodeling in spontaneously hypertensive rats

Evidence of sex-specific differences in renin-angiotensin-system (RAS) and arterial pressure has been shown in many mammals, including spontaneously hypertensive rats (SHRs). Although SHRs have been used extensively as a leading experimental model of hypertension, the effects of sex-specific differences in RAS on aortic function and related cardiac remodeling during aging and hypertension have not been documented in detail. We examined structural and functional changes in aorta and heart of female and male SHRs at the ages of 5, 14, 29, and 36 wk. SHRs of both sexes were hypertensive from 14 wk. Aortic endothelial dysfunction and fibrosis, left ventricular (LV) hypertrophy, and cardiac fibrosis were evident at the age of 29 wk in male SHRs but first appeared only at the age of 36 wk in female SHRs. There was a pronounced delay of matrix metalloproteinase-2 activity in the aorta and heart of female SHRs, which was associated with preservation of 40% more elastin and less extensive cardiac fibrosis than in males. At 5, 29, and 36 wk of age, female SHRs showed higher levels of aortic and myocardial AT2R and MasR mRNA and decreased ANG II-mediated aortic constriction. Although female SHRs had increased relaxation to AT2R stimulation at 5 and 29 wk compared with males, this difference disappeared at 36 wk of age. This study documents sex-specific differences in the temporal progression of aortic dysfunction and LV hypertrophy in SHRs, which are independent of arterial pressure and are apparently mediated by higher AT2R expression in the heart and aorta of female SHRs.