Acellular Human Placenta Small-Diameter Vessels as a Favorable Source of Super-Microsurgical Vascular Replacements: A Proof of Concept.

In this study, we aimed to evaluate the human placenta as a source of blood vessels that can be harvested for vascular graft fabrication in the submillimeter range. Our approach included graft modification to prevent thrombotic events. Submillimeter arterial grafts harvested from the human placenta were decellularized and chemically crosslinked to heparin. Graft performance was evaluated using a microsurgical arteriovenous loop (AVL) model in Lewis rats. Specimens were evaluated through hematoxylin-eosin and CD31 staining of histological sections to analyze host cell immigration and vascular remodeling. Graft patency was determined 3 weeks after implantation using a vascular patency test, histology, and micro-computed tomography. A total of 14 human placenta submillimeter vessel grafts were successfully decellularized and implanted into AVLs in rats. An appropriate inner diameter to graft length ratio of 0.81 ± 0.16 mm to 7.72 ± 3.20 mm was achieved in all animals. Grafts were left in situ for a mean of 24 ± 4 days. Decellularized human placental grafts had an overall patency rate of 71% and elicited no apparent immunological responses. Histological staining revealed host cell immigration into the graft and re-endothelialization of the vessel luminal surface. This study demonstrates that decellularized vascular grafts from the human placenta have the potential to serve as super-microsurgical vascular replacements.

Ethanol reduces the minimum alveolar concentration of sevoflurane in rats.

A high number of trauma patients are under the influence of alcohol. Since many of them need immediate surgical procedures, it is imperative to be aware of the interaction of alcohol with general anesthesia. To counter challenges that arise from clinical studies, we designed an animal experiment in which 48 adult Wistar rats either received 1 g · kg-1 ethanol, 2 g · kg-1 ethanol or placebo via intraperitoneal application. Subsequently, they were anesthetized with an individual concentration of sevoflurane. The minimum alveolar concentration (MAC) of the different groups was assessed using Dixon's up-and-down design and isotonic regression methods. The bootstrap estimate of the MAC of sevoflurane in the placebo group was 2.24 vol% (95% CI 1.97-2.94 vol%). In the low dose ethanol group, the bootstrap estimate was 1.65 vol% (95% CI 1.40-1.98 vol%), and in the high dose ethanol group, it was 1.08 vol% (95% CI 0.73-1.42 vol%). We therefore report that intraperitoneal application of 1 g · kg-1 or 2 g · kg-1 ethanol both resulted in a significant reduction of the MAC of sevoflurane in adult Wistar rats: by 26.3% and 51.8% respectively as compared to placebo.

The Role of Tenascin C in Cardiac Reverse Remodeling Following Banding-Debanding of the Ascending Aorta.

BACKGROUND: Tenascin-C (TN-C) plays a maladaptive role in left ventricular (LV) hypertrophy following pressure overload. However, the role of TN-C in LV regression following mechanical unloading is unknown. METHODS: LV hypertrophy was induced by transverse aortic constriction for 10 weeks followed by debanding for 2 weeks in wild type (Wt) and TN-C knockout (TN-C KO) mice. Cardiac function was assessed by serial magnetic resonance imaging. The expression of fibrotic markers and drivers (angiotensin-converting enzyme-1, ACE-1) was determined in LV tissue as well as human cardiac fibroblasts (HCFs) after TN-C treatment. RESULTS: Chronic pressure overload resulted in a significant decline in cardiac function associated with LV dilation as well as upregulation of TN-C, collagen 1 (Col 1), and ACE-1 in Wt as compared to TN-C KO mice. Reverse remodeling in Wt mice partially improved cardiac function and fibrotic marker expression; however, TN-C protein expression remained unchanged. In HCF, TN-C strongly induced the upregulation of ACE 1 and Col 1. CONCLUSIONS: Pressure overload, when lasting long enough to induce HF, has less potential for reverse remodeling in mice. This may be due to significant upregulation of TN-C expression, which stimulates ACE 1, Col 1, and alpha-smooth muscle actin (α-SMA) upregulation in fibroblasts. Consequently, addressing TN-C in LV hypertrophy might open a new window for future therapeutics.

Soluble guanylyl cyclase stimulation and phosphodiesterase-5 inhibition improve portal hypertension and reduce liver fibrosis in bile duct-ligated rats.

BACKGROUND: In cirrhosis, the nitric oxide-soluble guanylyl cyclase (sGC)-cyclic guanosine monophosphate (cGMP) pathway is impaired, which contributes to increased intrahepatic vascular resistance (IHVR) and fibrogenesis. We investigated if sGC stimulation (riociguat (RIO)), sGC activation (cinaciguat (CINA)) or phosphodiesterase (PDE)-5 inhibition (tadalafil (TADA)) improves portal hypertension (PHT) and liver fibrosis. METHODS: Fifty male Sprague-Dawley rats underwent bile-duct ligation (BDL) or sham operation. RIO (0.5 mg/kg), CINA (1 mg/kg), TADA (1.5 mg/kg) or vehicle (VEH) was administered from weeks 2 to 4 after BDL. At week 4, invasive haemodynamic measurements were performed, and liver fibrosis was assessed by histology (chromotrope-aniline blue (CAB), Picro-Sirius red (PSR)) and hepatic hydroxyproline content. RESULTS: Cirrhotic bile duct-ligated rats presented with PHT (13.1 ± 1.0 mmHg) and increased IHVR (4.9 ± 0.5 mmHg⋅min/mL). Both RIO (10.0 ± 0.7 mmHg, p = 0.021) and TADA (10.3 ± 0.9 mmHg, p = 0.050) decreased portal pressure by reducing IHVR (RIO: -41%, p = 0.005; TADA: -21%, p = 0.199) while not impacting heart rate, mean arterial pressure and portosystemic shunting. Hepatic cGMP levels increased upon RIO (+239%, p = 0.006) and TADA (+32%, p = 0.073) therapy. In contrast, CINA dosed at 1 mg/kg caused weight loss, arterial hypotension and hyperlactataemia in bile duct-ligated rats. Liver fibrosis area was significantly decreased by RIO (CAB: -48%, p = 0.011; PSR: -27%, p = 0.121) and TADA (CAB: -21%, p = 0.342; PSR: -52%, p = 0.013) compared to VEH-treated bile duct-ligated rats. Hepatic hydroxyproline content was reduced by RIO (from 503 ± 20 to 350 ± 30 µg/g, p = 0.003) and TADA (282 ± 50 µg/g, p = 0.003), in line with a reduction of the hepatic stellate cell activation markers smooth-muscle actin and phosphorylated moesin. Liver transaminases decreased under RIO (AST: -36%; ALT: -32%) and TADA (AST: -24%; ALT: -27%) treatment. Hepatic interleukin 6 gene expression was reduced in the RIO group (-56%, p = 0.053). CONCLUSION: In a rodent model of biliary cirrhosis, the sGC stimulator RIO and the PDE-5 inhibitor TADA improved PHT. The decrease of sinusoidal vascular resistance was paralleled by a reduction in liver fibrosis and hepatic inflammation, while systemic haemodynamics were not affected.

The soluble guanylate cyclase stimulator riociguat reduces fibrogenesis and portal pressure in cirrhotic rats.

In cirrhotic patients, portal hypertension (PHT) deteriorates survival, yet treatment options are limited. A major contributor to increased intrahepatic vasoconstriction in PHT is dysfunctional nitric-oxide signaling. Soluble guanylate cyclase (sGC) is the receptor of nitric-oxide and can be stimulated by riociguat. Riociguat is approved for pulmonary hypertension but has not been studied in liver cirrhosis. In this study we assessed the effects of riociguat on PHT and liver fibrosis in cholestatic (bile duct ligation, BDL) and toxic (carbon-tetrachloride, CCl4) rat models. In cirrhotic livers sGC expression was upregulated. In BDL rats, riociguat reduced liver fibrosis and decreased portal pressure without affecting systemic hemodynamics. In an early BDL disease stage, riociguat decreased bile duct proliferation, improved sinusoidal vascular dysfunction and inhibited angiogenesis. In advanced BDL riociguat exhibited anti-inflammatory effects. In CCl4 rats the beneficial effects of riociguat treatment were less pronounced and confined to an early disease stage. Similarly, in patients with cholestatic cirrhosis and PHT nitrates (that induce sGC activity) decreased portal pressure more effectively than in patients with non-cholestatic etiology. We also found an improvement of transaminases in patients with pulmonary hypertension receiving riociguat. Our findings support the clinical development of sGC stimulators in patients with cirrhotic PHT.

Myocardial infarct size measurement using geometric angle calculation.

BACKGROUND: In basic cardiovascular research focusing on animal models of myocardial infarction (MI), the measurement of infarct size is performed by planimetry of histological sections of the heart. However, in the setting of chronic MI with ongoing changes in ventricular geometry caused by wall thinning and hypertrophy, the scar area tends to become smaller. MATERIALS AND METHODS: Here, in this study we compared infarct measurements in tissue sections (of rat and porcine hearts) based on three different calculation approaches, that is, infarct area, infarct lengths and infarct angles utilizing the centroid of the left ventricle using a newly developed calculation approach. RESULTS: Infarct sizes from all three measurement approaches showed significant correlation with parameters of cardiac function. However, results derived from area measurements were significantly smaller than those obtained using the other two measurement approaches due to scar thinning (infarct size area: 14·81% ± 1·27 SEM, length: 23·94% ± 2·04 SEM, angle: 24·75% ± 2·13 SEM, P < 0·0001, n = 30). Moreover, results from angle measurements evidenced a much better correlation with parameters of cardiac function in a small animal model of chronic MI (e.g. ejection fraction, angle: r = -0·73; length: r = -0·64; area: r = -0·59, n = 30) as well as in a large animal model of acute MI (angle: r = -0·82; area: r = -0·67, n = 10). CONCLUSIONS: We concluded that area-, length- and angle-based measurements can be used to determine the relative infarct size in acute MI models, although an area-based measurement might be less accurate in the setting of chronic MI. Our new method of infarct angle measurement is a reliable and simple way to calculate infarct size compared with conventional measurement approaches.

Anti-thymocyte globulin induces neoangiogenesis and preserves cardiac function after experimental myocardial infarction.

RATIONALE: Acute myocardial infarction (AMI) followed by ventricular remodeling is the major cause of congestive heart failure and death in western world countries. OBJECTIVE: Of relevance are reports showing that infusion of apoptotic leucocytes or anti-lymphocyte serum after AMI reduces myocardial necrosis and preserves cardiac function. In order to corroborate this therapeutic mechanism, the utilization of an immunosuppressive agent with a comparable mechanism, such as anti-thymocyte globulin (ATG) was evaluated in this study. METHODS AND RESULTS: AMI was induced in rats by ligation of the left anterior descending artery. Initially after the onset of ischemia, rabbit ATG (10 mg/rat) was injected intravenously. In vitro and in vivo experiments showed that ATG induced a pronounced release of pro-angiogenic and chemotactic factors. Moreover, paracrine factors released from ATG co-incubated cell cultures conferred a down-regulation of p53 in cardiac myocytes. Rats that were injected with ATG evidenced higher numbers of CD68+ macrophages in the ischemic myocardium. Animals injected with ATG evidenced less myocardial necrosis, showed a significant reduction of infarct dimension and an improvement of post-AMI remodeling after six weeks (infarct dimension 24.9% vs. 11.4%, p<0.01). Moreover, a higher vessel density in the peri-infarct region indicated a better collateralization in rats that were injected with ATG. CONCLUSIONS: These data indicate that ATG, a therapeutic agent successfully applied in clinical transplant immunology, triggered cardioprotective effects after AMI that salvaged ischemic myocardium by down-regulation of p53. This might have raised the resistance against apoptotic cell death during ischemia. The combination of these mechanisms seems to be causative for improved cardiac function and less ventricular remodeling after experimental AMI.

Secretome of apoptotic peripheral blood cells (APOSEC) confers cytoprotection to cardiomyocytes and inhibits tissue remodelling after acute myocardial infarction: a preclinical study.

Heart failure following acute myocardial infarction (AMI) is a major cause of morbidity and mortality. Our previous observation that injection of apoptotic peripheral blood mononuclear cell (PBMC) suspensions was able to restore long-term cardiac function in a rat AMI model prompted us to study the effect of soluble factors derived from apoptotic PBMC on ventricular remodelling after AMI. Cell culture supernatants derived from irradiated apoptotic peripheral blood mononuclear cells (APOSEC) were collected and injected as a single dose intravenously after myocardial infarction in an experimental AMI rat model and in a porcine closed chest reperfused AMI model. Magnetic resonance imaging (MRI) and echocardiography were used to quantitate cardiac function. Analysis of soluble factors present in APOSEC was performed by enzyme-linked immunosorbent assay (ELISA) and activation of signalling cascades in human cardiomyocytes by APOSEC in vitro was studied by immunoblot analysis. Intravenous administration of a single dose of APOSEC resulted in a reduction of scar tissue formation in both AMI models. In the porcine reperfused AMI model, APOSEC led to higher values of ejection fraction (57.0 vs. 40.5%, p < 0.01), a better cardiac output (4.0 vs. 2.4 l/min, p < 0.001) and a reduced extent of infarction size (12.6 vs. 6.9%, p < 0.02) as determined by MRI. Exposure of primary human cardiac myocytes with APOSEC in vitro triggered the activation of pro-survival signalling-cascades (AKT, Erk1/2, CREB, c-Jun), increased anti-apoptotic gene products (Bcl-2, BAG1) and protected them from starvation-induced cell death. Intravenous infusion of culture supernatant of apoptotic PBMC attenuates myocardial remodelling in experimental AMI models. This effect is probably due to the activation of pro-survival signalling cascades in the affected cardiomyocytes.

Intravenous and intramyocardial injection of apoptotic white blood cell suspensions prevents ventricular remodelling by increasing elastin expression in cardiac scar tissue after myocardial infarction.

Congestive heart failure developing after acute myocardial infarction (AMI) is a major cause of morbidity and mortality. Clinical trials of cell-based therapy after AMI evidenced only a moderate benefit. We could show previously that suspensions of apoptotic peripheral blood mononuclear cells (PBMC) are able to reduce myocardial damage in a rat model of AMI. Here we experimentally examined the biochemical mechanisms involved in preventing ventricular remodelling and preserving cardiac function after AMI. Cell suspensions of apoptotic cells were injected intravenously or intramyocardially after experimental AMI induced by coronary artery ligation in rats. Administration of cell culture medium or viable PBMC served as controls. Immunohistological analysis was performed to analyse the cellular infiltrate in the ischaemic myocardium. Cardiac function was quantified by echocardiography. Planimetry of the infarcted hearts showed a significant reduction of infarction size and an improvement of post AMI remodelling in rats treated with suspensions of apoptotic PBMC (injected either intravenously or intramoycardially). Moreover, these hearts evidenced enhanced homing of macrophages and cells staining positive for c-kit, FLK-1, IGF-I and FGF-2 as compared to controls. A major finding in this study further was that the ratio of elastic and collagenous fibres within the scar tissue was altered in a favourable fashion in rats injected with apoptotic cells. Intravenous or intramyocardial injection of apoptotic cell suspensions results in attenuation of myocardial remodelling after experimental AMI, preserves left ventricular function, increases homing of regenerative cells and alters the composition of cardiac scar tissue. The higher expression of elastic fibres provides passive energy to the cardiac scar tissue and results in prevention of ventricular remodelling.