Mast Cells in the Microenvironment of Hepatocellular Carcinoma Confer Favorable Prognosis: A Retrospective Study using QuPath Image Analysis Software.

The insights provided by in-situ detection of immune cells within hepatocellular carcinoma (HCC) might present information on patient outcomes. Studies investigating the expression and localization of immune cells within tumor tissues are associated with several challenges, including a lack of precise annotation for tumor regions and random selection of microscopic fields of view. QuPath is an open-source, user-friendly software that could meet the growing need for digital pathology in whole-slide image (WSI) analysis. The infiltration of HCC and adjacent tissues by CD1a+ immature dendritic cells (iDCs), CD117+ mast cells, and NKp46+ natural killer cells (NKs) cells was assessed immunohistochemically in representative specimens of 67 patients with HCC who underwent curative resection. The area fraction (AF) of positively stained cells was assessed automatically in WSIs using QuPath in the tumor center (TC), inner margin (IM), outer margin (OM), and peritumor (PT) area. The prognostic significance of immune cells was evaluated for time to recurrence (TTR), disease-free survival (DFS), and overall survival (OS). The AF of mast cells was significantly greater than the AF of NKs, and the AF of iDCs was significantly lower compared to NKs in each region of interest. High AFs of mast cells in the IM and PT areas were associated with longer DFS. In addition, high AF of mast cells in IM was associated with longer OS. Computer-assisted analysis using this software is a suitable tool for obtaining prognostic information for tumor-infiltrating immune cells (iDCs, mast cells, and NKs) in different regions of HCC after resection. Mast cells displayed the greatest AF in all regions of interest (ROIs). Mast cells in the peritumor region and IM showed a positive prognostic significance.

Prognostic role of macrophages and mast cells in the microenvironment of hepatocellular carcinoma after resection.

BACKGROUND: The prognostic significance of mast cells and different phenotypes of macrophages in the microenvironment of hepatocellular carcinoma (HCC) following resection is unclear. We aimed in this study to assess the local distribution of infiltrating macrophages and mast cells of specific phenotypes in tissues of HCC and to evaluate their prognostic values for survival of post-surgical patients. METHODS: The clinicopathological and follow-up data of 70 patients with HCC, who underwent curative resection of tumor from 1997 to 2019, were collected. The infiltration of CD68+ and CD163+ macrophages and CD117+ mast cells was assessed immunohistochemically in representative resected specimens of HCC and adjacent tissues. The area fraction (AF) of positively stained cells was estimated automatically using QuPath image analysis software in several regions, such as tumor center (TC), inner margin (IM), outer margin (OM), and peritumor (PT) area. The prognostic significance of immune cells, individually and in associations, for time to recurrence (TTR), disease-free survival (DFS), and overall survival (OS) was evaluated using Kaplan-Meier and Cox regression analyses. RESULTS: High AF of CD68+ macrophages in TC and IM and high AF of mast cells in IM and PT area were associated with a longer DFS. High AF of CD163+ macrophages in PT area correlated with a shorter DFS. Patients from CD163TChigh & CD68TClow group had a shorter DFS compared to all the rest of the groups, and cases with CD163IMlow & CD68IMhigh demonstrated significantly longer DFS compared to low AF of both markers. Patients from CD68IMhigh & CD163PTlow group, CD117IMhigh & CD163PTlow group, and CD117PThigh & CD163PTlow group had a significantly longer DFS compared to all other combinations of respective cells. CONCLUSIONS: The individual prognostic impact of CD68+ and CD163+ macrophages and mast cells in the microenvironment of HCC after resection depends on their abundance and location, whereas the cumulative impact is built upon combination of different cell phenotypes within and between regions.

Protein expression and localization of ABC transporters in pancreatic adenocarcinoma: Prognostic role of ABCC8.

BACKGROUND: ATP-binding cassette (ABC) transporters translocate various substances across cellular membranes. Their deregulation may cause cancer drug resistance or perturbations in the supply of building blocks for cancer cells and modify patients' prognosis. This study investigated protein expression and cellular localization of the previously suggested putative prognostic biomarkers - ABCB2/TAP1, ABCC7/CFTR, ABCC8/SUR1, and ABCD4 in patients with pancreatic ductal adenocarcinoma (PDAC). METHODS: Protein expression and localization were assessed by immunohistochemistry in formalin-fixed paraffin-embedded primary tumor tissue blocks of 61 PDAC patients and associated with clinical data and the survival of patients. RESULTS: No CFTR protein expression was observed in PDAC, while TAP1 and ABCC8 were expressed predominantly in the cytoplasm of tumor cells. Most samples (81 %) had detectable both membranous and cytoplasmic ABCD4 staining and 42 % had ABCD4 expressed in the apical orientation. Negative membranous ABCD4 staining was significantly more frequent in advanced stage III or IV tumors (p = 0.022). Small or medium counts of individual ABCC8-positive cells in the stroma surrounding tumor tubules were also more often found in stage III or IV (p = 0.044). Patients with moderate or strong ABCC8 cytoplasmic staining intensity in tumor cells had a 3.5-fold higher risk of disease progression than those with weak staining (p = 0.002). CONCLUSIONS: The study shows for the first time that the cytoplasmic ABCC8 protein expression has prognostic value in PDAC.

Exome Sequencing of Paired Colorectal Carcinomas and Synchronous Liver Metastases for Prognosis and Therapy Prediction.

PURPOSE: Analysis of somatic variant profiles in retrospectively collected pairs of primary tumors and synchronous liver metastases from surgically treated patients with colorectal carcinomas. Mutational profiles were compared between groups of patients stratified by response to chemotherapy and survival. PATIENTS AND METHODS: The study used whole-exome sequencing of tumor sample pairs from 20 patients diagnosed and treated at a single center. The Cancer Genome Atlas COAD-READ data set (n = 380) was used for validation in silico, where possible. RESULTS: The most frequently altered oncodrivers were APC (55% in primaries and 60% in metastases), TP53 (50/45), TRIP11 (30/5), FAT4 (20/25), and KRAS (15/25). Harboring variants with a high or moderate predicted functional effect in KRAS in primary tumors was significantly associated with poor relapse-free survival in both our sample set and the validation data set. We found a number of additional prognostic associations, including mutational load, alterations in individual genes, oncodriver pathways, and single base substitution (SBS) signatures in primary tissues, which were not confirmed by validation. Altered ATM, DNAH11, and MUC5AC, or a higher share of SBS24 signature in metastases seemed to represent poor prognostic factors, but because of a lack of suitable validation data sets, these results must be treated with extreme caution. No gene or profile was significantly associated with response to chemotherapy. CONCLUSION: Taken together, we report subtle differences in exome mutational profiles between paired primary tumors and synchronous liver metastases and a distinct prognostic relevance of KRAS in primary tumors. Although the general scarcity of primary tumor-synchronous metastasis sample pairs with high-quality clinical data makes robust validation difficult, this study provides potentially valuable data for utilization in precision oncology and could serve as a springboard for larger studies.

Histological mapping of healing of the small and large intestine - A quantitative study in a porcine model.

BACKGROUND: Gastrointestinal anastomoses are performed in many patients every year. The pathogenesis of aberrant anastomotic healing and the causes of intestinal leakage are not fully understood. The present study gathered and critically evaluated histological quantitative data to deepen current knowledge of anastomotic healing in the small and large intestine and its complications and outline the options for further experimental in vivo research in large porcine animal models. METHODS: Three groups of porcine intestinal anastomoses were compared: small intestine without defect (SI; n = 7), small intestine with an additional defect (SID; n = 8), and large intestine (LI; n = 7). Multilevel sampling (2112 micrographs) and stereological methods were used for histological quantification of proliferation (Ki-67 immunohistochemistry), neutrophil infiltration (myeloperoxidase staining), vascularity (von Willebrand factor) and type I and type III collagen formation (picrosirius red in polarized light) within the region of anastomosis compared to the region outside of anastomosis. RESULTS: Quantitative histological evaluation revealed the following results. i) Proliferation, vascularity, and collagen, but not neutrophils, were more highly expressed within the anastomosis than outside of the anastomosis region. ii) Porcine large and small intestine were not interchangeable based on histological evaluation of surgical experiments. The presence or absence of an additional experimental defect strongly affected healing, but the healing seemed complete after 21 days. iii) The microscopic structure of small intestine segments was more affected by their proximity to the anastomosis than the structure of large intestine segments. CONCLUSIONS: Histological quantification was more laborious than the previously used semiquantitative scoring system evaluating the healing rate of intestinal anastomoses, but it provided detailed maps of biological processes within individual intestine layers. The primary data collected in the study are open and available for power sample analyses to calculate the minimum numbers of samples justified in future experiments on porcine intestines. The porcine intestine is a promising animal model with translational potential for human surgery.

CTNNB1 mutations, TERT polymorphism and CD8+ cell densities in resected hepatocellular carcinoma are associated with longer time to recurrence.

BACKGROUND: Hepatocellular carcinoma (HCC) is a fatal disease characterized by early genetic alterations in telomerase reverse transcriptase promoter (TERTp) and ß-catenin (CTNNB1) genes and immune cell activation in the tumor microenvironment. As a novel approach, we wanted to assess patient survival influenced by combined presence of mutations and densities of CD8+ cytotoxic T cells. METHODS: Tissue samples were obtained from 67 HCC patients who had undergone resection. We analysed CD8+ T cells density, TERTp mutations, rs2853669 polymorphism, and CTNNB1 mutations. These variables were evaluated for time to recurrence (TTR) and disease free survival (DFS). RESULTS: TERTp mutations were found in 75.8% and CTNNB1 mutations in 35.6% of the patients. TERTp mutations were not associated with survival but polymorphism rs2853669 in TERTp was associated with improved TTR and DFS. CTNNB1 mutations were associated with improving TTR. High density of CD8+ T-lymphocytes in tumor center and invasive margin correlated with longer TTR and DFS. Combined genetic and immune factors further improved survival showing higher predictive values. E.g., combining CTNNB1 mutations and high density of CD8+ T-lymphocytes in tumor center yielded HRs of 0.12 (0.03-0.52), p = 0.005 for TTR and 0.25 (0.09-0.74), p = 0.01 for DFS. CONCLUSION: The results outline a novel integrative approach for prognostication through combining independent predictive factors from genetic and immune cell profiles. However, larger studies are needed to explore multiple cell types in the tumor microenvironment.

Decellularization of Porcine Carotid Arteries: From the Vessel to the High-Quality Scaffold in Five Hours.

The use of biologically derived vessels as small-diameter vascular grafts in vascular diseases is currently intensely studied. Vessel decellularization provides a biocompatible scaffold with very low immunogenicity that avoids immunosuppression after transplantation. Good scaffold preservation is important as it facilitates successful cell repopulation. In addition, mechanical characteristics have to be carefully evaluated when the graft is intended to be used as an artery due to the high pressures the vessel is subjected to. Here, we present a new and fast decellularization protocol for porcine carotid arteries, followed by investigation of the quality of obtained vessel scaffolds in terms of maintenance of important extracellular matrix components, mechanical resistance, and compatibility with human endothelial cells. Our results evidence that our decellularization protocol minimally alters both the presence of scaffold proteins and their mechanical behavior and human endothelial cells could adhere to the scaffold in vitro. We conclude that if a suitable protocol is used, a high-quality decellularized arterial scaffold of non-human origin can be promptly obtained, having a great potential to be recellularized and used as an arterial graft in transplantation medicine.

T- and B-Cells in the Inner Invasive Margin of Hepatocellular Carcinoma after Resection Associate with Favorable Prognosis.

In this retrospective study on 67 patients with hepatocellular carcinoma (HCC), after tumor resection, we evaluated the significance of CD3+ and CD8+ T-lymphocytes and CD20+ B-lymphocytes in tumor and non-tumor liver for time to recurrence (TTR), disease-free survival (DFS) and overall survival. After immunohistochemical staining, the density of nucleated lymphocyte profiles (QA) was estimated stereologically in the tumor center (TC), inner margin (inn M), outer margin (out M), peritumor and non-tumor liver. In TC, intermediate and high QA of CD8+ cells predicted longer TTR, whereas CD3+ and CD20+ were predictive only at high QA. DFS was predicted by high QA of CD3+, CD8+ and CD20+ cells in TC. The inn M harbored smaller QA of CD3+, CD8+ and CD20+ lymphocytes than out M. In contrast to out M, high T-cells' QA and intermediate and high B-cell QA in inn M predicted longer TTR and DFS. High inn M/out M QA ratios of CD3+ and CD20+ cells were associated with longer TTR and DFS, whereas high inn M/out M QA ratio of CD8+ was predictive only for DFS. Patients with intermediate-high QA of combined CD8+ and CD20+ cells in inn M showed longer TTR and DFS, compared to CD8+-high or CD20+-high alone. Our findings highlight overall heterogeneity of the tumor invasive margin, the importance of inn M, and the predictive role of B-cells.

Using virtual microscopy for the development of sampling strategies in quantitative histology and design-based stereology.

Only a fraction of specimens under study are usually selected for quantification in histology. Multilevel sampling or tissue probes, slides and fields of view (FOVs) in the regions of interest (ROIs) are required. In general, all parts of the organs under study should be given the same probability to be taken into account; that is, the sampling should be unbiased on all levels. The objective of our study was to provide an overview of the use of virtual microscopy in the context of developing sampling strategies of FOVs for stereological quantification. We elaborated this idea on 18 examples from multiple fields of histology, including quantification of extracellular matrix and muscle tissue, quantification of organ and tumour microvessels and tumour-infiltrating lymphocytes, assessing osseointegration of bone implants, healing of intestine anastomoses and osteochondral defects, counting brain neurons, counting nuclei in vitro cell cultures and others. We provided practical implications for the most common situations, such as exhaustive sampling of ROIs, sampling ROIs of different sizes, sampling the same ROIs for multiple histological methods, sampling more ROIs with variable intensities or using various objectives, multistage sampling and virtual sampling. Recommendations were provided for pilot studies on systematic uniform random sampling of FOVs as a part of optimizing the efficiency of histological quantification to prevent over- or undersampling. We critically discussed the pros and cons of using virtual sections for sampling FOVs from whole scanned sections. Our review demonstrated that whole slide scans of histological sections facilitate the design of sampling strategies for quantitative histology.

Decellularized xenogeneic scaffolds in transplantation and tissue engineering: Immunogenicity versus positive cell stimulation.

Seriously compromised function of some organs can only be restored by transplantation. Due to the shortage of human donors, the need to find another source of organs is of primary importance. Decellularized scaffolds of non-human origin are being studied as highly potential biomaterials for tissue engineering. Their biological nature and thus the ability to provide a naturally-derived environment for human cells to adhere and grow highlights their great advantage in comparison to synthetic scaffolds. Nevertheless, since every biomaterial implanted in the body generates immune reaction, studying the interaction of the scaffold with the surrounding tissues is necessary. This review aims to summarize current knowledge on the immunogenicity of semi-xenografts involved in transplantation. Moreover, positive aspects of the interaction between xenogeneic scaffold and human cells are discussed, focusing on specific roles of proteins associated with extracellular matrix in cell adhesion and signalling.

Double-layered Nanofibrous Patch for Prevention of Anastomotic Leakage and Peritoneal Adhesions, Experimental Study.

BACKGROUND/AIM: Anastomotic leakage is a feared complication in colorectal surgery. Postoperative peritoneal adhesions can also cause life-threatening conditions. Nanofibrous materials showed their pro-healing properties in various studies. The aim of the study was to evaluate the impact of double-layered nanofibrous materials on anastomotic healing and peritoneal adhesions formation. MATERIALS AND METHODS: Two versions of double-layered materials from polycaprolactone and polyvinyl alcohol were applied on defective anastomosis on the small intestine of healthy pigs. The control group remained with uncovered defect. Tissue specimens were subjected to histological analysis and adhesion scoring after 3 weeks of observation. RESULTS: The wound healing was inferior in the experimental groups, however, no anastomotic leakage was observed and the applied material always kept covering the defect. The extent of adhesions was larger in the experimental groups. CONCLUSION: Nanofibrous materials may prevent anastomotic leakage but delay healing.

Reinforcement of Colonic Anastomosis with Improved Ultrafine Nanofibrous Patch: Experiment on Pig.

Anastomotic leakage is a dreadful complication in colorectal surgery. It has a negative impact on postoperative mortality, long term life quality and oncological results. Nanofibrous polycaprolactone materials have shown pro-healing properties in various applications before. Our team developed several versions of these for healing support of colorectal anastomoses with promising results in previous years. In this study, we developed highly porous biocompatible polycaprolactone nanofibrous patches. We constructed a defective anastomosis on the large intestine of 16 pigs, covered the anastomoses with the patch in 8 animals (Experimental group) and left the rest uncovered (Control group). After 21 days of observation we evaluated postoperative changes, signs of leakage and other complications. The samples were assessed histologically according to standardized protocols. The material was easy to work with. All animals survived with no major complication. There were no differences in intestinal wall integrity between the groups and there were no signs of anastomotic leakage in any animal. The levels of collagen were significantly higher in the Experimental group, which we consider to be an indirect sign of higher mechanical strength. The material shall be further perfected in the future and possibly combined with active molecules to specifically influence the healing process.

Semantic Segmentation of Intralobular and Extralobular Tissue from Liver Scaffold H&E Images.

Decellularized tissue is an important source for biological tissue engineering. Evaluation of the quality of decellularized tissue is performed using scanned images of hematoxylin-eosin stained (H&E) tissue sections and is usually dependent on the observer. The first step in creating a tool for the assessment of the quality of the liver scaffold without observer bias is the automatic segmentation of the whole slide image into three classes: the background, intralobular area, and extralobular area. Such segmentation enables to perform the texture analysis in the intralobular area of the liver scaffold, which is crucial part in the recellularization procedure. Existing semi-automatic methods for general segmentation (i.e., thresholding, watershed, etc.) do not meet the quality requirements. Moreover, there are no methods available to solve this task automatically. Given the low amount of training data, we proposed a two-stage method. The first stage is based on classification of simple hand-crafted descriptors of the pixels and their neighborhoods. This method is trained on partially annotated data. Its outputs are used for training of the second-stage approach, which is based on a convolutional neural network (CNN). Our architecture inspired by U-Net reaches very promising results, despite a very low amount of the training data. We provide qualitative and quantitative data for both stages. With the best training setup, we reach 90.70% recognition accuracy.

Novel morphological multi-scale evaluation system for quality assessment of decellularized liver scaffolds.

Decellularized scaffolds can serve as an excellent three-dimensional environment for cell repopulation. They maintain tissue-specific microarchitecture of extracellular matrix proteins with important spatial cues for cell adhesion, migration, growth, and differentiation. However, criteria for quality assessment of the three-dimensional structure of decellularized scaffolds are rather fragmented, usually study-specific, and mostly semi-quantitative. Thus, we aimed to develop a robust structural assessment system for decellularized porcine liver scaffolds. Five scaffolds of different quality were used to establish the new evaluation system. We combined conventional semi-quantitative scoring criteria with a quantitative scaffold evaluation based on automated image analysis. For the quantitation, we developed a specific open source software tool (ScaffAn) applying algorithms designed for texture analysis, segmentation, and skeletonization. ScaffAn calculates selected parameters characterizing structural features of porcine liver scaffolds such as the sinusoidal network. After evaluating individual scaffolds, the total scores predicted scaffold interaction with cells in terms of cell adhesion. Higher scores corresponded to higher numbers of cells attached to the scaffolds. Moreover, our analysis revealed that the conventional system could not identify fine differences between good quality scaffolds while the additional use of ScaffAn allowed discrimination. This led us to the conclusion that only using the combined score resulted in the best discrimination between different quality scaffolds. Overall, our newly defined evaluation system has the potential to select the liver scaffolds most suitable for recellularization, and can represent a step toward better success in liver tissue engineering.

Experimental fortification of intestinal anastomoses with nanofibrous materials in a large animal model.

Anastomotic leakage is a severe complication in gastrointestinal surgery. It is often a reason for reoperation together with intestinal passage blockage due to formation of peritoneal adhesions. Different materials as local prevention of these complications have been studied, none of which are nowadays routinely used in clinical practice. Nanofabrics created proved to promote healing with their structure similar to extracellular matrix. We decided to study their impact on anastomotic healing and formation of peritoneal adhesions. We performed an experiment on 24 piglets. We constructed 3 hand sutured end-to-end anastomoses on the small intestine of each pig. We covered the anastomoses with a sheet of polycaprolactone nanomaterial in the first experimental group, with a sheet of copolymer of polylactic acid with polycaprolactone in the second one and no fortifying material was used in the Control group. The animals were sacrificed after 3 weeks of observation. Clinical, biochemical and macroscopic signs of anastomotic leakage or intestinal obstruction were monitored, the quality of the scar tissue was assessed histologically, and a newly developed scoring system was employed to evaluate the presence of adhesions. The material is easy to manipulate with. There was no mortality or major morbidity in our groups. No statistical difference was found inbetween the groups in the matter of level of peritoneal adhesions or the quality of the anastomoses. We created a new adhesion scoring system. The material appears to be safe however needs to be studied further to prove its' positive effects.

Protein expression of ABCC2 and SLC22A3 associates with prognosis of pancreatic adenocarcinoma.

ATP-binding cassette (ABC) and solute carrier (SLC) transporters translocate diverse substances across cellular membranes and their deregulation may cause drug resistance of cancers. This study investigated significance of protein expression and cellular localization of the previously suggested putative prognostic markers ABCC2 and SLC22A3 in pancreatic cancer patients. Protein localization and brush border staining intensity of ABCC2 and SLC22A3 was assessed in tumor tissue blocks of 65 pancreatic cancer patients and associated with clinical data and survival of patients with regard to therapy. Negative SLC22A3 brush border staining in pancreatic tumors significantly increased the risk of both disease progression and patient´s death in univariate analyses. Multivariate analyses confirmed the association of SLC22A3 expression with progression-free survival of patients. A subgroup analysis of patients treated with regimens based on nucleoside analogs suggested that patients with negative brush border staining or apical localization of SLC22A3 in tumor cells have worse overall survival. The combination of positive ABCC2 and negative SLC22A3 brush border staining predicted worst overall survival and patients with positive brush border staining of both proteins had best overall and progression-free survival. The present study shows for the first time that the protein presence and to some extent also localization of SLC22A3 significantly associate with prognosis of pancreatic cancer in both unstratified and chemotherapy-treated patients. The combination of ABCC2 and SLC22A3 brush border staining also needs further attention in this regard.

The exaggerated salt-sensitive response in hypertensive transgenic rats (TGR mRen-2) fostered by a normotensive female.

Suboptimal conditions during prenatal and early postnatal development can increase risk of hypertension later in life. We studied consequences of a changed perinatal environment by initiating the cross-fostering of homozygous Ren-2 transgenic rat (TGR) offspring to normotensive, transgene-negative control mothers, and vice versa. We hypothesized that cross-fostering to a normotensive female can attenuate the development of malignant hypertension in TGR offspring (TGRx) and change their salt-sensitive response. Blood pressure (BP) was monitored by the telemetry system under normal salt intake, and BP responses to increased salt intake in the phase of established hypertension. Under normal salt conditions, BP was not markedly different in cross-fostered animals compared with controls. However, BP responses to 2% salt intake led to a stronger BP response in TGRx during the active phase when compared with the control TGR group. The TGRx also exhibited increased albuminuria, lower sodium excretion, and creatinine clearance under higher salt intake compared with control salt intake. Higher salt intake resulted in a significant increase of aldosterone concentrations only in the TGRx group; moreover, TGRx rats exhibited more pronounced renal injury compared with controls. In conclusion, our data indicate that cross-fostering in TGR not only did not attenuate the development of hypertension but, on the contrary, led to the deterioration of BP regulation, particularly due to exaggerated salt sensitivity and sodium retention in TGRx. Results underline the important role of the mother during lactation in postnatal development of the offspring, since these changes reflected different ion content in milk of a particular strain of rats.

20-Hydroxyeicosatetraenoic acid antagonist attenuates the development of malignant hypertension and reverses it once established: a study in Cyp1a1-Ren-2 transgenic rats.

We hypothesized that vascular actions of 20-hydroxyeicosatetraenoic acid (20-HETE), the product of cytochrome P450 (CYP450)-dependent ω-hydroxylase, potentiate prohypertensive actions of angiotensin II (ANG II) in Cyp1a1-Ren-2 transgenic rats, a model of ANG II-dependent malignant hypertension. Therefore, we evaluated the antihypertensive effectiveness of 20-HETE receptor antagonist (AAA) in this model. Malignant hypertension was induced in Cyp1a1-Ren-2 transgenic rats by activation of the renin gene using indole-3-carbinol (I3C), a natural xenobiotic. Treatment with AAA was started either simultaneously with induction of hypertension or 10 days later, during established hypertension. Systolic blood pressure (SBP) was monitored by radiotelemetry, indices of renal and cardiac injury, and kidney ANG II levels were determined. In I3C-induced hypertensive rats, early AAA treatment reduced SBP elevation (to 161 ± 3 compared with 199 ± 3 mmHg in untreated I3C-induced rats), reduced albuminuria, glomerulosclerosis index, and cardiac hypertrophy (P<0.05 in all cases). Untreated I3C-induced rats showed augmented kidney ANG II (405 ± 14 compared with 52 ± 3 fmol/g in non-induced rats, P<0.05) which was markedly lowered by AAA treatment (72 ± 6 fmol/g). Remarkably, in TGR with established hypertension, AAA also decreased SBP (from 187 ± 4 to 158 ± 4 mmHg, P<0.05) and exhibited organoprotective effects in addition to marked suppression of kidney ANG II levels. In conclusion, 20-HETE antagonist attenuated the development and largely reversed the established ANG II-dependent malignant hypertension, likely via suppression of intrarenal ANG II levels. This suggests that intrarenal ANG II activation by 20-HETE is important in the pathophysiology of this hypertension form.

Isovolumic loading of the failing heart by intraventricular placement of a spring expander attenuates cardiac atrophy after heterotopic heart transplantation.

Cardiac atrophy is the most common complication of prolonged application of the left ventricle (LV) assist device (LVAD) in patients with advanced heart failure (HF). Our aim was to evaluate the course of unloading-induced cardiac atrophy in rats with failing hearts, and to examine if increased isovolumic loading obtained by intraventricular implantation of an especially designed spring expander would attenuate this process. Heterotopic abdominal heart transplantation (HTx) was used as a rat model of heart unloading. HF was induced by volume overload achieved by creation of the aorto-caval fistula (ACF). The degree of cardiac atrophy was assessed as the weight ratio of the heterotopically transplanted heart (HW) to the control heart. Isovolumic loading was increased by intraventricular implantation of a stainless steel three-branch spring expander. The course of cardiac atrophy was evaluated on days 7, 14, 21, and 28 after HTx Seven days unloading by HTx in failing hearts sufficed to substantially decrease the HW (-59 ± 3%), the decrease progressed when measured on days 14, 21, and 28 after HTx Implantation of the spring expander significantly reduced the decreases in whole HW at all the time points (-39 ± 3 compared with -59 ± 3, -52 ± 2 compared with -69 ± 3, -51 ± 2 compared with -71 ± 2, and -44 ± 2 compared with -71 ± 3%, respectively; P<0.05 in each case). We conclude that the enhanced isovolumic heart loading obtained by implantation of the spring expander attenuates the development of unloading-induced cardiac atrophy in the failing rat heart.

Renin-angiotensin system blockade alone or combined with ETA receptor blockade: effects on the course of chronic kidney disease in 5/6 nephrectomized Ren-2 transgenic hypertensive rats.

BACKGROUND: Early addition of endothelin (ET) type A (ETA) receptor blockade to complex renin-angiotensin system (RAS) blockade has previously been shown to provide better renoprotection against progression of chronic kidney disease (CKD) in Ren-2 transgenic hypertensive rats (TGR) after 5/6 renal ablation (5/6 NX). In this study, we examined if additional protection is provided when ETA blockade is applied in rats with already developed CKD. METHODS: For complex RAS inhibition, an angiotensin-converting enzyme inhibitor along with angiotensin II type 1 receptor blocker was used. Alternatively, ETA receptor blocker was added to the RAS blockade. The treatments were initiated 6 weeks after 5/6 NX and the follow-up period was 50 weeks. RESULTS: When applied in established CKD, addition of ETA receptor blockade to the complex RAS blockade brought no further improvement of the survival rate (30% in both groups); surprisingly, aggravated albuminuria (588 ± 47 vs. 245 ± 38 mg/24 h, p < 0.05) did not reduce renal glomerular injury index (1.25 ± 0.29 vs. 1.44 ± 0.26), did not prevent the decrease in creatinine clearance (203 ± 21 vs. 253 ± 17 µl/min/100 g body weight), and did not attenuate cardiac hypertrophy to a greater extent than observed in 5/6 NX TGR treated with complex RAS blockade alone. CONCLUSIONS: When applied in the advanced phase of CKD, addition of ETA receptor blockade to the complex RAS blockade brings no further beneficial renoprotective effects on the CKD progression in 5/6 NX TGR, in addition to those seen with RAS blockade alone.