Different Notch signaling in cells from calcified bicuspid and tricuspid aortic valves.

AIMS: Calcific aortic valve disease is the most common heart valve disease in the Western world. Bicuspid and tricuspid aortic valve calcifications are traditionally considered together although the dynamics of the disease progression is different between the two groups of patients. Notch signaling is critical for bicuspid valve development and NOTCH1 mutations are associated with bicuspid valve and calcification. We hypothesized that Notch-dependent mechanisms of valve mineralization might be different in the two groups. METHODS AND RESULTS: We used aortic valve interstitial cells and valve endothelial cells from patients with calcific aortic stenosis with bicuspid or tricuspid aortic valve. Expression of Notch-related genes in valve interstitial cells by qPCR was different between bicuspid and tricuspid groups. Discriminant analysis of gene expression pattern in the interstitial cells revealed that the cells from calcified bicuspid valves formed a separate group from calcified tricuspid and control cells. Interstitial cells from bicuspid calcified valves demonstrated significantly higher sensitivity to stimuli at early stages of induced proosteogenic differentiation and were significantly more sensitive to the activation of proosteogenic OPN, ALP and POSTIN expression by Notch activation. Notch-activated endothelial-to-mesenchymal transition and the corresponding expression of HEY1 and SLUG were also more prominent in bicuspid valve derived endothelial cells compared to the cells from calcified tricuspid and healthy valves. CONCLUSION: Early signaling events including Notch-dependent mechanisms that are responsible for the initiation of aortic valve calcification are different between the patients with bicuspid and tricuspid aortic valves.

Mitochondrial DNA damage and repair during ischemia-reperfusion injury of the heart.

Ischemia-reperfusion (IR) injury of the heart generates reactive oxygen species that oxidize macromolecules including mitochondrial DNA (mtDNA). The 8-oxoguanine DNA glycosylase (OGG1) works synergistically with MutY DNA glycosylase (MYH) to maintain mtDNA integrity. Our objective was to study the functional outcome of lacking the repair enzymes OGG1 and MYH after myocardial IR and we hypothesized that OGG1 and MYH are important enzymes to preserve mtDNA and heart function after IR. Ex vivo global ischemia for 30min followed by 10min of reperfusion induced mtDNA damage that was removed within 60min of reperfusion in wild-type mice. After 60min of reperfusion the ogg1(-/-) mice demonstrated increased mtDNA copy number and decreased mtDNA damage removal suggesting that OGG1 is responsible for removal of IR-induced mtDNA damage and copy number regulation. mtDNA damage was not detected in the ogg1(-/-)/myh(-/-), inferring that adenine opposite 8-oxoguanine is an abundant mtDNA lesion upon IR. The level and integrity of mtDNA were restored in all genotypes after 35min of regional ischemia and six week reperfusion with no change in cardiac function. No consistent upregulation of other mitochondrial base excision repair enzymes in any of our knockout models was found. Thus repair of mtDNA oxidative base lesions may not be important for maintenance of cardiac function during IR injury in vivo. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease."

Per-unit-living tissue normalization of real-time RT-PCR data in ischemic rat hearts.

In studies of gene expression in acute ischemic heart tissue, internal reference genes need to show stable expression per-unit-living tissue to hinder dead cells from biasing real-time RT-PCR data. Until now, this important issue has not been appropriately investigated. We hypothesized that the expression of seven internal reference genes would show stable per-unit-living tissue expression in Langendorff-perfused rat hearts subjected to ischemia-reperfusion. This was found for cyclophilin A, GAPDH, RPL-32, and PolR2A mRNA, with GAPDH showing the highest degree of stability (R = 0.11), suggesting unchanged rates of mRNA transcription in live cells and complete degradation of mRNA from dead cells. The infarct size-dependent degradation of GAPDH was further supported by a close correlation between changes in GAPDH mRNA and changes in RNA quality measured as RNA integrity number (R = 0.90, P < 0.05). In contrast, ß-actin and 18S rRNA showed stable expression per-unit-weight tissue and a positive correlation with infarct size (R = 0.61 and R = 0.77, P < 0.05 for both analyses). The amount of total RNA extracted per-unit-weight tissue did not differ between groups despite wide variation in infarct size (7.1-50.1%). When ß-actin expression was assessed using four different normalization strategies, GAPDH and geNorm provided appropriate per-unit-living expression, while 18S and total RNA resulted in marked underestimations. In studies of ischemic tissues, we recommend using geometric averaging of carefully selected reference genes for normalization of real-time RT-PCR data. A marked shift in the mRNA/rRNA ratio renders rRNA as useless for normalization purposes.

Mesenchymal stromal cells fail to alleviate experimental graft-versus-host disease in rats transplanted with major histocompatibility complex-mismatched bone marrow.

Mesenchymal stromal cells (MSC) can be used to treat graft-versus-host disease (GVHD) caused by allogeneic stem cell transplantation (allo-SCT). The effectiveness of this therapy has been variable in clinical trials and in experimental animal models. In this study, we investigated the ability of bone marrow (BM)-derived MSC to alleviate GVHD in an experimental rat model of allo-SCT using two different combinations of major histocompatibility complex (MHC) mismatch with survival as the primary endpoint. Recipient rats received total body irradiation and a transplant of T cell-depleted donor BM cells with either a full [PVG.7B → BN] or a partial MHC mismatch [PVG.1U → PVG.R23] restricted to the class II and non-classical class I sub-regions (RT1-B/D-CE/N/M). GVHD was invoked by infusion of graded doses of donor leukocytes 2 weeks after allo-SCT. Weekly doses of MSC were injected starting on the day of donor leukocyte infusion. No significant overall improvement of mortality and morbidity was observed in the two transplantation settings. Stimulation of MSC with exogenous tumor necrosis factor α and interferon (IFN)γ prior to infusion could not rescue BM-transplanted rats from lethal acute GVHD. In conclusion, repeated administrations of MSC failed to alleviate GVHD after fully or partially MHC-mismatched allo-SCT in the rat.

Intracellular Complement Component 3 Attenuated Ischemia-Reperfusion Injury in the Isolated Buffer-Perfused Mouse Heart and Is Associated With Improved Metabolic Homeostasis.

The innate immune system is rapidly activated during myocardial infarction and blockade of extracellular complement system reduces infarct size. Intracellular complement, however, appears to be closely linked to metabolic pathways and its role in ischemia-reperfusion injury is unknown and may be different from complement activation in the circulation. The purpose of the present study was to investigate the role of intracellular complement in isolated, retrogradely buffer-perfused hearts and cardiac cells from adult male wild type mice (WT) and from adult male mice with knockout of complement component 3 (C3KO). Main findings: (i) Intracellular C3 protein was expressed in isolated cardiomyocytes and in whole hearts, (ii) after ischemia-reperfusion injury, C3KO hearts had larger infarct size (32 ± 9% in C3KO vs. 22 ± 7% in WT; p=0.008) and impaired post-ischemic relaxation compared to WT hearts, (iii) C3KO cardiomyocytes had lower basal oxidative respiration compared to WT cardiomyocytes, (iv) blocking mTOR decreased Akt phosphorylation in WT, but not in C3KO cardiomyocytes, (v) after ischemia, WT hearts had higher levels of ATP, but lower levels of both reduced and oxidized nicotinamide adenine dinucleotide (NADH and NAD+, respectively) compared to C3KO hearts. Conclusion: intracellular C3 protected the heart against ischemia-reperfusion injury, possibly due to its role in metabolic pathways important for energy production and cell survival.

Natural killer cell subsets in man and rodents.

NK cells are important contributors to the early immune defence against infected or transformed cells. They are rapidly activated in response to cytokines, whereby they exert their effector functions. NK cell responses are controlled by a multitude of receptors, which are expressed by subpopulations of NK cells with distinct phenotypes and functionalities. Direct comparisons between species are often difficult because of differences in the expression of NK cell receptors and other markers. In addition, NK cells change their phenotype and effector functions during differentiation, by tissue-specific factors, or upon activation, complicating interpretations. We will here review the similarities and differences between the major NK cell subsets in man and two well-characterized rodent models.

Control of rat natural killer cell-mediated allorecognition by a major histocompatibility complex region encoding nonclassical class I antigens.

The ability of natural killer (NK) cells to eliminate normal allogeneic hemic cells is well established in several species including mice, rats, and humans. The controlling elements for NK susceptibility in these species map to the major histocompatibility complex (MHC), but in contrast to findings in mice and humans, the mode of inheritance is not always recessive in rats. This finding is not easily explained by the missing self and hemopoietic histocompatibility (Hh) models for NK recognition, and has led to the idea that certain alloantigens may trigger NK cell reactivity. In our in vitro system for assessing rat NK alloreactivity, we have employed target and inhibitor cells from a large panel of MHC congenic, intra-MHC recombinant and MHC mutant rat strains, as well as appropriate F1 hybrids between them, and we show the following: (a) The nonclassical class I (RT1.C) region was most important in determining the susceptibility of target cells to alloreactive NK cells in vitro. Lymphocyte susceptibility to lysis in vivo also mapped to the C region, which supports the concept that the in vivo and in vitro alloreactivity assays reflect the same recognition process. (b) Four different RT1-controlled NK allospecificities (represented by the u, l, a, and n haplotypes) could be discerned when we used polyclonal NK cells from the PVG (RT1c) strain as effector cells. Three of the target specificities recognized were controlled mainly by the RT1.C region. (c) The expression of RT1.C region-controlled parental strain NK allodeterminants could be demonstrated in F1 hybrids heterozygous for the C region alone and were therefore inherited nonrecessively. (d) Loss of an RT1.C region-controlled NK allospecificity could be shown with the MHC mutant LEW.1LM1 rat strain characterized by a genomic deletion of about 100 kb of the C region. Taken together, these observations have demonstrated a major importance of the nonclassical class I region, i.e., RT1.C, in controlling rat NK allorecognition, and have thereby assigned a hitherto undescribed immunological property to this region. Furthermore, some of the present data are consistent with the existence of polymorphic NK-triggering alloantigens that are coded for by the RT1.C region.

Molecular cloning of the cDNA encoding pp36, a tyrosine-phosphorylated adaptor protein selectively expressed by T cells and natural killer cells.

Activation of T and natural killer (NK) cells leads to the tyrosine phosphorylation of pp36 and to its association with several signaling molecules, including phospholipase Cgamma-1 and Grb2. Microsequencing of peptides derived from purified rat pp36 protein led to the cloning, in rat and man, of cDNA encoding a T- and NK cell-specific protein with several putative Src homology 2 domain-binding motifs. A rabbit antiserum directed against a peptide sequence from the cloned rat molecule recognized tyrosine phosphorylated pp36 from pervanadate-treated rat thymocytes. When expressed in 293T human fibroblast cells and tyrosine-phosphorylated, pp36 associated with phospholipase Cgamma-1 and Grb2. Studies with GST-Grb2 fusion proteins demonstrated that the association was specific for the Src homology 2 domain of Grb-2. Molecular cloning of the gene encoding pp36 should facilitate studies examining the role of this adaptor protein in proximal signaling events during T and NK cell activation.

SNF472, a novel anti-crystallization agent, inhibits induced calcification in an in vitro model of human aortic valve calcification.

The purpose of the present study was to investigate whether SNF472, the hexasodium salt of myo-inositol hexaphosphate (IP6 or phytate): 1. Inhibits induced calcification in cultured aortic valve interstitial cells (VIC) as an in vitro model of aortic valve stenosis and 2. Whether inhibition is different in VIC obtained from healthy and calcified aortic valves. VIC from healthy (n = 5) and calcified (n = 7) human aortic valves were seeded in basic growth medium, osteogenic differentiation medium alone, or in osteogenic medium with SNF472 (3, 10, and 30 µM) and cultivated for 3 weeks. Calcification was quantified spectrophotometrically after Alizarin Red staining. In VIC from calcified valves, a complete inhibition of calcification was observed with SNF472 concentrations of 10 and 30 µM (p < .01), significantly stronger than in VIC from healthy valves. When SNF472 was added to VIC after 1 week in osteogenic medium, 30 and 100 µM SNF472 inhibited the progression of ongoing calcification by 81 and 100% (p < .01), respectively. The same concentrations of SNF472 given after 2 weeks reduced calcification by 35 and 40% respectively (not significant). SNF472 inhibited both the formation and the progression of calcification with the strongest effect in VIC from calcified valves.

Mode of perfusion influences infarct size, coronary flow and stress kinases in the isolated mouse heart.

AIM: The isolated, retrogradely perfused heart (modified Langendorff model) is a widely used method in experimental heart research. The presence of an intraventricular balloon is necessary to get functional measurements. We have previously shown that the balloon induces phosphorylation of some suggested cardioprotective mitogen-activated protein kinases (MAPK): P38-MAPK, ERK 1/2 and JNK. We hypothesized that the balloon could influence cardioprotection, protect against ischaemia reperfusion injury and interfere with coronary flow. METHODS AND RESULTS: Isolated mouse hearts were perfused for 5, 10, 20, 40 and 60 min with a balloon in the left ventricle. We found a wavelike phosphorylation of all MAPK while AKT displayed a gradual dephosphorylation when compared to non-perfused hearts. Hearts were subjected to 20 min of stabilization with or without the balloon, followed by 35 min of ischaemia and 120 min of reperfusion. Although the MAPK were phosphorylated, the infarcts were larger in the balloon group. When the balloon was present during the entire protocol, compared to removal at the end of ischaemia, the infarct size was also larger, especially in the endocardial layer. The balloon reduced post-ischaemic endocardial coronary flow, despite a higher average flow, indicating a hyperperfused epicard. Blocking the balloon-induced ERK 1/2 phosphorylation during stabilization did not affect infarct size. The effect of post-conditioning was influenced by the balloon, showing reduced infarct size when the balloon was present. CONCLUSION: The balloon used for pressure measurements may contributes to cell death possibly by reducing endocardial coronary flow.

Relationship between tumor growth characteristics and preferential sites of growth.

The anterior-posterior incidence of spontaneous mammary tumor development and the anterior-posterior gradient of growth potential among transplanted tumors was studied in C3H/He and C3Hf/He mice. The relative incidence of spontaneous tumor development in the five mammary gland pairs showed no anterior-posterior bias, but it was in proportion to the quantity of tissue in the individual mammary glands. The transplantability and growth rate of 164 spontaneous C3H/He and 67 spontaneous C3Hf/He mammary carcinomas were tested and compared in anterior and posterior subcutaneous sites and at mammary implantation sites. Initially, in their early transplant generations, most subcutaneous tumor implants (67%) grew significantly better near the shoulder than in an implantation site near the hip. At the same time, implants from the same tumor tissue grew equally well in anterior (#2) and posterior (#4) mammary glands. Without exception, all transplanted tumors grew better in a mammary gland than at a subcutaneous site. Some tumors (12%) that initially would grow only in mammary glands gained subcutaneous transplantability with increased growth rate. With increasing growth rate, the tumors' anterior subcutaneous growth preference decreased. Anterior subcutaneous growth preference was not related to immunologic tumor characteristics. Implants of slow-growing tumors grew better in the anterior subcutaneous implantation site where the greater blood flow improved their growth conditions and survival rate.

Plasma cell infiltrate of the primary tumor as a source of early systemic protection against metastases in mice.

Histologic examination of sc implants of the syngeneic C3H/He mammary carcinoma MC2 showed that the accumulation of a large number of lymphoid cells in the stroma around the implants in C3H/He mice was a constant feature of the early primary host response. This stromal reaction had two main components, the first composed of small lymphocytes and the second composed of larger blast cells with a high proportion of plasma cells. The surgical removal of a 6-day-old tumor implant with its surrounding stromal reaction, if performed 4 days before systemic dissemination of MC2 cells via injection into the left ventricle, resulted in a more frequent growth of the disseminated cells as compared to the frequency of growth in mice carrying their tumor implants. This increased frequency of growth in surgically cured mice could be reduced by repeated transfusions of plasma from MC2-bearing hosts. Repeated ip injections of antigen, in the form of 2 X 10(5) inactivated tumor cells after tumor excision, did not support the development of strong systemic resistance against metastases. When sublethal whole-body radiation was given before the sc tumor implant, the stromal reaction was much reduced, and the surgical removal of sc tumor implants from irradiated mice resulted in only a minor increase in the growth of tumor cells injected into the circulation compared to the growth of tumor cells injected into irradiated mice carrying their tumor implants. It appears that a strong, local primary immune reaction may act as a temporary accessory lymphoid organ, constituting an early and potent source of systemic immune protective factors.

Long-term cyclophosphamide treatments against primary mammary tumors in C3H/He mice.

Tumor-free inbred female C3H/He mice were given weekly injections of cyclophosphamide to prevent or delay the expected occurrence of spontaneous mammary carcinomas. Chemotherapy was started at an age when the mice would already have developed preneoplastic hyperplastic alveolar nodules and tumors were likely to appear within a few weeks. Treatments were given for periods ranging from 10 to 50 weeks with various schedules and doses. The mice were observed for the development of tumors until they died or were killed. Tumors were excised as they appeared. Treatments were most effective in reduction of the number of primary tumors when started early and given continuously. Longer term, low-dose treatments gave better results than short-term, high-dose treatments, although the total dose given was the same. The prophylactic effect of the drug appeared to be by the destruction of occult, drug-sensitive tumors, rather than by delay of their appearance. The toxicity of moderate, continuous drug administration was well tolerated with no mortality and only minor transient weight loss.

Effects of liposome-entrapped doxorubicin on liver metastases of mouse colon carcinomas 26 and 38.

The effects of doxorubicin (DOX) and DOX entrapped in standardized liposomes [mean diameter, 0.15 micron; (DOX-Lip)] on the survival of mice bearing liver metastases of mouse colon carcinoma CT38LD (C57BL/6J mice) or CT26 (BALB/c mice) were investigated. In vitro cultured CT38LD cells were more sensitive to DOX than CT26. In vivo DOX and DOX-Lip, administered iv 10 mg/kg weekly to a maximum of five injections, increased the life-spans of mice bearing CT38LD liver metastases 32% (P less than .05) and 64% (P less than .05), respectively. DOX-Lip was more effective than DOX in prolonging survival (P less than .05). Free DOX did not significantly increase the life-spans of mice bearing CT26 liver metastases (P greater than .5), whereas DOX-Lip increased the life-spans 35% (P less than .05). The results suggest that liposomal delivery of agents to the liver can enhance therapeutic activity and could be used as an arm of protocols for adjuvant therapy of liver metastases.

Local and systemic effects during interleukin-2 therapy of mouse mammary tumors.

The therapeutic effects of 12 daily peritumor injections of from 100 to 300,000 units of recombinant human interleukin-2 were tested against the syngeneic, immunogenic mammary carcinoma MC2 implanted s.c. into C3H/He mice. Local therapeutic effect on injected tumors was observed down to 300 units of interleukin-2 per injection. Cures of injected tumors were obtained with 1,000 units and more per injection. Systemic therapeutic effect on contralateral, uninjected tumors in treated mice was discernible at 5,000 units and more per injection. Hepatic periportal cellular swelling with mononuclear infiltration, and renal tubular edema were observed at 7,000 units or more per injection. Hepatic and renal repairs were rapid and complete with 50,000 units and less per injection. Hepatic necrosis developed above 50,000 units per injection. Deaths resulted from 100,000 units and more per injection. It is concluded that interleukin-2 can be a safe and effective therapeutic agent at a wide range of doses well below those that may be expected to have serious negative side effects.

Local interleukin 2 therapy of mouse mammary tumors of various immunogenicities.

The local and systemic therapeutic effects of multiple injections of recombinant human interleukin 2 (IL-2), at doses of 5,000 to 100,000 units each, were tested against intramammary implants of 12 syngeneic C3H mouse mammary tumors of various immunogenicities. Among 5 tumors with transplantation-type immunogenicity and with mononuclear leukocyte infiltration, 4 tumors were affected by IL-2 therapy. A fast-growing, moderately immunogenic tumor was not affected. Among 7 tumors which did not display transplantation-type immunogenicity, 4 tumors did, nevertheless, attract mononuclear leukocytes. Among these 4 tumors, the 2 slowest growing tumors were affected by IL-2 therapy. Local and systemic therapeutic effects resulted from peritumor IL-2 injections, but not from injections 2 cm from a tumor. The results indicate that the therapeutic effectiveness of IL-2 depends on the ability of a tumor to attract IL-2 responsive immune effector cells and that therapeutic effectiveness is reduced by faster tumor growth.

Occurrence of shared nonviral tumor-associated transplantation-type antigens among C3H/He mammary carcinomas.

The occurrence of shared nonviral tumor-associated antigens among 18 immunogenic C3H/He mammary carcinomas was investigated in vivo. By cross-immunization and s.c. challenge between pairs of tumors in 37 different combinations, six of the tumors were found to cross-immunize with one to three other tumors. A minimum of three different antigenic specificities were found to be shared among cross-reacting tumors.

Inherent changes in the in vivo growth characteristics of C3H/He mammary carcinomas.

The ability of C3H/He mammary carcinoma cells to grow in the lungs after i.v. injection was repeatedly tested with cells from tumors which were kept in serial s.c. passage in syngeneic female mice. The s.c. growth rate and the s.c. transplantation immunogenicity were also determined for each transplant generation. The ability of a tumor to grow in the lungs, which appeared in most tumors only after repeated s.c. passages, coincided mainly with increased growth rate and not with the loss of immunogenicity and/or gain of endogenous growth-stimulating factors. In each combination of cross-reactivity tested, transplantation immunogenicity was tumor specific, and growth stimulation was not tumor specific. Three of ten tumors were retested in serial passages started again from pieces of the primary tumors stored in liquid N2, and the identical changes recurred in the same, or in nearly the same, transplant generations. This indicates that certain variable neoplastic characteristics may be inherent and will appear not haphazardly, but according to a genetically predetermined schedule.

Mammary tumor immune enhancement in mice by local hyperemia.

The mammary carcinoma MC2 causes a strong immune response in syngeneic, female C3H/He mice, and growing s.c. implants will regress spontaneously in about 20% of untreated hosts. Hyperemia is part of the local immune reaction against MC2 implants. An accelerated local hyperemic reaction at MC2 implants could be created in normal mice by the i.p. injection of blood or plasma from MC2 hosts at an early (Day 17) stage of immunization. In these mice given injections, MC2 growth was enhanced, and the incidence of spontaneous regressions was reduced. Local hyperemia caused by heat also promoted the growth of MC2 implants. In contrast, injections of blood or plasma from MC2 hosts at an advanced (Day 35) stage of immunization reduced the growth of MC2 implants and increased the incidence of their spontaneous regression. It is concluded that increased blood supply to a tumor in the absence of adequate systemic immunity favors tumor growth, and that this represents a new, additional mechanism in the immune enhancement phenomenon.

Changing transplantation characteristics with serial in vivo passage of C3H/He mammary carcinomas.

The transplantation immunogenicity of spontaneous C3H/He mammary carcinomas was studied by means of the surgery-challenge procedure during serial in vivo passages in syngeneic mice. Reciprocal cross-sensitization and challenge tests between late transplant generations and early transplant generations (from liquid N2 storage) of the same tumors showed that factors responsible for transplantation resistance and factors causing stimulated tumor growth were present in the tumors at the same time as independent variables. The immunogenicity and the immunosensitivity of tumors were seen as dependent variables. The relative prominence of the characteristics of immunogenicity and growth stimulation changed with continuous in vivo passages. Transplantation immunogenicity was tumor specific in four of five tumors tested. Growth stimulation was, in each of four combinations tested, not tumor specific.