Cancer risk and mortality after solid organ transplantation: A population-based 30-year cohort study in Finland.

Cancer is a significant cause of morbidity and mortality after solid organ transplantation (SOT) and related to lifelong immunosuppression. This retrospective registry study assessed for the first time in Finland population-based cancer risk and cancer mortality after all SOTs (lung and childhood transplantations included) as standardized incidence ratios (SIRs) and standardized mortality ratios (SMRs). Data from transplant registries were linked with the data of Finnish Cancer Registry and Statistics Finland. We followed 6548 consecutive first SOT recipients from 1 January 1987 to 31 December 2016 translating to 66 741 person-years (median follow-up time 8.9 years [interquartile range 4.0-15.1]). In total, 2096 cancers were found in 1483 patients (23% of all patients). Majority of cancers (53%) were nonmelanoma skin cancers (NMSCs). The overall SIR was 3.6 (95% confidence interval [CI]: 3.5-3.8) and the SIR excluding NMSCs was 2.2 (95% CI: 2.0-2.3). SIR for all cancers was highest for heart (5.0) and lowest for liver (2.7) recipients. Most common cancer types after NMSCs were non-Hodgkin lymphoma (NHL), SIR 9.9 (95% CI: 8.5-11.4) and kidney cancer, SIR 7.3 (95% CI: 6.0-8.8). Cancer-related deaths were 17% (n = 408) of all deaths after first month post transplantation. SMR for all cancers was 2.5 (95% CI: 2.2-2.7) and for NHL 13.6 (95% CI: 10.7-16.8). Notably, overall SIR for cancer was lower in later period (2000-2016), 3.0 (95% CI: 2.8-3.2), than in earlier period (1987-1999), 4.3 (95% CI: 4.0-4.5), P < .001. Decrease in cancer incidence was temporally associated with major changes in immunosuppression in the 2000s.

Outcome after heart-lung or lung transplantation in patients with Eisenmenger syndrome.

OBJECTIVE: The optimal timing for transplantation is unclear in patients with Eisenmenger syndrome (ES). We investigated post-transplantation survival and transplantation-specific morbidity after heart-lung transplantation (HLTx) or lung transplantation (LTx) in a cohort of Nordic patients with ES to aid decision-making for scheduling transplantation. METHODS: We performed a retrospective, descriptive, population-based study of patients with ES who underwent transplantation from 1985 to 2012. RESULTS: Among 714 patients with ES in the Nordic region, 63 (9%) underwent transplantation. The median age at transplantation was 31.9 (IQR 21.1-42.3) years. Within 30 days after transplantation, seven patients (11%) died. The median survival was 12.0 (95% CI 7.6 to 16.4) years and the overall 1-year, 5-year, 10-year and 15-year survival rates were 84.1%, 69.7%, 55.8% and 40.6%, respectively. For patients alive 1 year post-transplantation, the median conditional survival was 14.8 years (95% CI 8.0 to 21.8), with 5-year, 10-year and 15-year survival rates of 83.3%, 67.2% and 50.0%, respectively. There was no difference in median survival after HLTx (n=57) and LTx (n=6) (14.9 vs 10.6 years, p=0.718). Median cardiac allograft vasculopathy, bronchiolitis obliterans syndrome and dialysis/kidney transplantation-free survival rates were 11.2 (95% CI 7.8 to 14.6), 6.9 (95% CI 2.6 to 11.1) and 11.2 (95% CI 8.8 to 13.7) years, respectively. The leading causes of death after the perioperative period were infection (36.7%), bronchiolitis obliterans syndrome (23.3%) and heart failure (13.3%). CONCLUSIONS: This study shows that satisfactory post-transplantation survival, comparable with contemporary HTx and LTx data, without severe comorbidities such as cardiac allograft vasculopathy, bronchiolitis obliterans syndrome and dialysis, is achievable in patients with ES, with a conditional survival of nearly 15 years.

Characterization of different fat depots in NAFLD using inflammation-associated proteome, lipidome and metabolome.

Non-alcoholic fatty liver disease (NAFLD) is recognized as a liver manifestation of metabolic syndrome, accompanied with excessive fat accumulation in the liver and other vital organs. Ectopic fat accumulation was previously associated with negative effects at the systemic and local level in the human body. Thus, we aimed to identify and assess the predictive capability of novel potential metabolic biomarkers for ectopic fat depots in non-diabetic men with NAFLD, using the inflammation-associated proteome, lipidome and metabolome. Myocardial and hepatic triglycerides were measured with magnetic spectroscopy while function of left ventricle, pericardial and epicardial fat, subcutaneous and visceral adipose tissue were measured with magnetic resonance imaging. Measured ectopic fat depots were profiled and predicted using a Random Forest algorithm, and by estimating the Area Under the Receiver Operating Characteristic curves. We have identified distinct metabolic signatures of fat depots in the liver (TAG50:1, glutamate, diSM18:0 and CE20:3), pericardium (N-palmitoyl-sphinganine, HGF, diSM18:0, glutamate, and TNFSF14), epicardium (sphingomyelin, CE20:3, PC38:3 and TNFSF14), and myocardium (CE20:3, LAPTGF-ß1, glutamate and glucose). Our analyses highlighted non-invasive biomarkers that accurately predict ectopic fat depots, and reflect their distinct metabolic signatures in subjects with NAFLD.

Ectopic fat depots and left ventricular function in nondiabetic men with nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease has emerged as a novel cardiovascular risk factor. The aim of the study was to assess the effect of different ectopic fat depots on left ventricular (LV) function in subjects with nonalcoholic fatty liver disease. METHODS AND RESULTS: Myocardial and hepatic triglyceride contents were measured with 1.5 T magnetic resonance spectroscopy and LV function, visceral adipose tissue (VAT) and subcutaneous adipose tissue, epicardial and pericardial fat by MRI in 75 nondiabetic men. Subjects were stratified by hepatic triglyceride content into low, moderate, and high liver fat groups. Myocardial triglyceride, epicardial and pericardial fat, VAT, and subcutaneous adipose tissue increased stepwise from low to high liver fat group. Parameters of LV diastolic function showed a stepwise decrease over tertiles of liver fat and VAT, and they were inversely correlated with hepatic triglyceride, VAT, and VAT/subcutaneous adipose tissue ratio. In multivariable analyses, hepatic triglyceride and VAT were independent predictors of LV diastolic function, whereas myocardial triglyceride was not associated with measures of diastolic function. CONCLUSIONS: Myocardial triglyceride, epicardial and pericardial fat increased with increasing amount of liver fat and VAT. Hepatic steatosis and VAT associated with significant changes in LV structure and function. The association of LV diastolic function with hepatic triglyceride and VAT may be because of toxic systemic effects. The effects of myocardial triglyceride on LV structure and function seem to be more complex than previously thought and merit further study.

Cardiac steatosis in patients with dilated cardiomyopathy.

OBJECTIVE: Ectopic fat accumulation within and around the heart has been related to increased risk of heart disease. Limited data exist on cardiac adiposity in subjects with dilated cardiomyopathy (DCM). The aim of the study was to examine the components of cardiac steatosis and their relationship to LV structure and function in non-diabetic DCM patients. METHODS: Myocardial and hepatic triglyceride (TG) contents were measured with 1.5 T magnetic resonance spectroscopy (MRS), and LV function, visceral adipose (VAT) and abdominal subcutaneous tissue (SAT), epicardial and pericardial fat by MRI in 10 non-diabetic men with DCM and in 20 controls. RESULTS: In face of comparable intra-abdominal fat depots, myocardial TG [0.41% (0.21-2.19) vs. 0.86% (0.31-2.24), p=0.038] was markedly lower and epicardial (895 mm2±110 vs. 664 mm2±180, p=0.002) and pericardial fat [2173 mm2 (616-3673) vs 1168 mm2 (266-2319), p=0.039] depots were larger in patients with DCM compared with controls. In subjects with DCM, the LV global function index was decreased to a greater extent than the LV EF [21%±6 vs. 34% (16-40)]. CONCLUSIONS: Myocardial TG content decreased and epicardial and pericardial fat depots increased in non-diabetic subjects with DCM. Although recognised as a site of ectopic fat accumulation, the derangement of myocardial TG seems to play a specific role in the myocardial energy metabolism in congestive heart failure.

Cardiac steatosis and left ventricular function in men with metabolic syndrome.

BACKGROUND: Ectopic accumulation of fat accompanies visceral obesity with detrimental effects. Lipid oversupply to cardiomyocytes leads to cardiac steatosis, and in animal studies lipotoxicity has been associated with impaired left ventricular (LV) function. In humans, studies have yielded inconclusive results. The aim of the study was to evaluate the role of epicardial, pericardial and myocardial fat depots on LV structure and function in male subjects with metabolic syndrome (MetS). METHODS: A study population of 37 men with MetS and 38 men without MetS underwent cardiovascular magnetic resonance and proton magnetic spectroscopy at 1.5 T to assess LV function, epicardial and pericardial fat area and myocardial triglyceride (TG) content. RESULTS: All three fat deposits were greater in the MetS than in the control group (p <0.001). LV diastolic dysfunction was associated with MetS as measured by absolute (471 mL/s vs. 667 mL/s, p = 0.002) and normalized (3.37 s⁻¹ vs. 3.75 s⁻¹, p = 0.02) LV early diastolic peak filling rate and the ratio of early diastole (68% vs. 78%, p = 0.001). The amount of epicardial and pericardial fat correlated inversely with LV diastolic function. However, myocardial TG content was not independently associated with LV diastolic dysfunction. CONCLUSIONS: In MetS, accumulation of epicardial and pericardial fat is linked to the severity of structural and functional alterations of the heart. The role of increased intramyocardial TG in MetS is more complex and merits further study.

Cardiac steatosis associates with visceral obesity in nondiabetic obese men.

BACKGROUND: Liver fat and visceral adiposity are involved in the development of the metabolic syndrome (MetS). Ectopic fat accumulation within and around the heart has been related to increased risk of heart disease. The aim of this study was to explore components of cardiac steatosis and their relationship to intra-abdominal ectopic fat deposits and cardiometabolic risk factors in nondiabetic obese men. METHODS: Myocardial and hepatic triglyceride (TG) contents were measured with 1.5 T magnetic resonance spectroscopy, and visceral adipose (VAT), abdominal subcutaneous tissue (SAT), epicardial and pericardial fat by magnetic resonance imaging in 37 men with the MetS and in 40 men without the MetS. RESULTS: Myocardial and hepatic TG contents, VAT, SAT, epicardial fat volumes, and pericardial fat volumes were higher in men with the MetS compared with subjects without the MetS (P < .001). All components of cardiac steatosis correlated with SAT, VAT, and hepatic TG content and the correlations seemed to be strongest with VAT. Myocardial TG content, epicardial fat, pericardial fat, VAT, and hepatic TG content correlated with waist circumference, body mass index, high-density lipoprotein cholesterol TGs, very low-density lipoprotein-1 TGs, and the insulin-resistance homeostasis model assessment index. VAT was a predictor of TGs, high-density lipoprotein cholesterol, and measures of glucose metabolism, whereas age and SAT were determinants of blood pressure parameters. CONCLUSIONS: We suggest that visceral obesity is the best predictor of epicardial and pericardial fat in abdominally obese subjects. Myocardial TG content may present a separate entity that is influenced by factors beyond visceral adiposity.

Mast cells promote atherosclerosis by inducing both an atherogenic lipid profile and vascular inflammation.

Accumulating in vitro and in vivo studies have proposed a role for mast cells in the pathogenesis of atherosclerosis. Here, we studied the role of mast cells in lipoprotein metabolism, a key element in the atherosclerotic disease. Male mice deficient in low-density lipoprotein receptors and mast cells on a Western diet for 26 weeks had significantly less atherosclerotic changes both in aortic sinus (55%, P = 0.0009) and in aorta (31%, P = 0.049), as compared to mast cell-competent littermates. Mast cell-deficient female mice had significantly less atherosclerotic changes in aortic sinus (43%, P = 0.011). Furthermore, we found a significant positive correlation between the extent of atherosclerosis and the number of adventitial/perivascular mast cells in aortic sinus of mast cell-competent mice (r = 0.615, P = 0.015). Serum cholesterol and triglyceride levels were significantly lower in both male (63%, P = 0.0005 and 57%, P = 0.004) and female (73%, P = 0.00009 and 54%, P = 0.007) mast cell-deficient mice, with a concomitant decrease in atherogenic apoB-containing particles and serum prebeta-high-density lipoprotein and phospholipid transfer protein activity in both male (69% and 24%) and female (74% and 54%) mast cell-deficient mice. Serum soluble intercellular adhesion molecule was decreased in both male (32%, P = 0.004) and female (28%, P = 0.003) mast cell-deficient mice, whereas serum amyloid A was similar between mast cell-deficient and competent mice. In conclusion, mast cells participate in the pathogenesis of atherosclerosis in ldlr(-/-) mice by inducing both an atherogenic lipid profile and vascular inflammation.

Function and regulation of the complement system in cardiovascular diseases.

The complement system plays a central role in innate immunity and also regulates adaptive immunity. The complement system has been demonstrated to contribute to various diseases, including cardiovascular diseases. Complement is extensively activated in atherosclerotic lesions, in arterial aneurysms, and in the myocardium of ischemic and failing hearts. Accumulating evidence shows that limitation of excessive complement activation under these conditions may hold therapeutic value. On the other hand, defects in the classical complement pathway predispose to vasculitis and atherosclerosis, possibly due to ineffective clearance of apoptotic/necrotic cells and abnormal processing of immune complexes. Here, we describe complement activation and regulation in cardiovascular diseases and discuss the evidence derived mainly from experimental animals suggesting that modulation of complement activation may alter the course of these disorders.

Receptors for the anaphylatoxins C3a and C5a are expressed in human atherosclerotic coronary plaques.

The anaphylatoxins C3a and C5a are potent chemotactic and pro-inflammatory peptides that are released during complement activation, and recent clinical work have suggested them a role in acute coronary events. Here we studied whether human coronary plaques express anaphylatoxin receptors C3aR and C5aR, i.e. whether they have the potential to respond to anaphylatoxins. For this purpose, both normal (n=14) and atherosclerotic (n=20) human coronary artery samples were collected for histological and PCR analyses. Immunohistochemistry demonstrated that in atherosclerotic, but not in normal intimas, C3aR and C5aR were present. Consistently, PCR analysis showed that the expression of both receptors was >5-fold increased in the atherosclerotic plaques (p<0.01). Double immunofluorescence stainings revealed that in the plaques the principal cells expressing both C3aR and C5aR were macrophages. Moreover, T cells expressed C5aR and a small fraction of them also expressed C3aR, the mast cells expressed C5aR, whereas endothelial cells and subendothelial smooth muscle cells expressed both C3aR and C5aR. In conclusion, the presence of receptors for anaphylatoxins in human coronary plaques suggests that anaphylatoxins activate coronary plaques, and points the complement system as a potential therapeutic target in attempts to stabilize them.

Complement regulation in human atherosclerotic coronary lesions. Immunohistochemical evidence that C4b-binding protein negatively regulates the classical complement pathway, and that C5b-9 is formed via the alternative complement pathway.

OBJECTIVE: The complement system is activated in human atherosclerotic lesions and may hence aggravate local inflammation. We studied the presence and localization of C4b-binding protein (C4bp), the major inhibitor of the classical complement pathway, in human atherosclerotic lesions in relation to complement activation products and protein S, which circulates in complex with C4bp. METHODS AND RESULTS: Immunohistochemistry of human coronary arteries showed C4bp to be virtually absent in normal arteries but present in early and advanced atherosclerotic lesions. In the lesions, C4bp is associated with proteoglycans, and affinity chromatography showed that C4bp interacts with human arterial proteoglycans. Areas containing C4bp also contained IgM and C4 suggesting that C4bp is involved in the regulation of the classical complement pathway. However, C5b-9 was virtually absent in these areas but, instead, colocalized with properdin deeper in the intima, suggesting that C5b-9 is formed by the alternative complement pathway. A fraction of C4bp was associated with protein S and apoptotic cells. CONCLUSIONS: The results indicate that C4bp regulates the classical complement pathway in human atherosclerotic lesions. Thus, unlike the alternative pathway, the classical complement pathway does not generate C5b-9, but is likely to be involved in the clean-up of apoptotic cells and cell debris in the arterial intima.

Low-density lipoprotein modified by macrophage-derived lysosomal hydrolases induces expression and secretion of IL-8 via p38 MAPK and NF-kappaB by human monocyte-derived macrophages.

OBJECTIVE: Modified lipoproteins induce inflammatory reactions in the atherosclerotic arterial wall. We have previously found that macrophages in atherosclerotic lesions secrete lysosomal hydrolases that can modify low-density-lipoprotein (LDL) in vitro to generate "hydrolase-modified LDL" (H-LDL). Here, we studied whether H-LDL exerts inflammatory effects on cultured human macrophages. METHODS AND RESULTS: Using cytokine cDNA arrays, we found that H-LDL induced expression of IL-8, but not of the anti-inflammatory cytokines IL-10 and transforming growth factor (TGF)-beta, in human monocyte-derived macrophages. H-LDL induced rapid phosphorylation of the p38 mitogen-activated protein kinase (MAPK), nuclear translocation of 2 transcription factors, nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1), and time-dependent secretion of IL-8 from the macrophages. Inhibition of MAPKs and of transcription factors showed that p38 MAPK and NF-kappaB, but not ERK1/2, JNK, or AP-1, were crucial for the H-LDL-induced IL-8 secretion from the macrophages. CONCLUSIONS: The results show that by activating p38 MAPK and NF-kappaB, macrophage hydrolases modify LDL into biologically active particles capable of triggering the secretion of IL-8 in macrophages. Thus, activated hydrolase-secreting macrophages in atherosclerotic lesions may sustain a proatherogenic extracellular environment by hydrolyzing LDL and triggering it to act in an autocrine or paracrine fashion to induce IL-8 secretion by the plaque macrophages.

OxLDL-IgG immune complexes induce survival of human monocytes.

OBJECTIVE: Immune complexes containing oxidatively modified low-density lipoprotein (oxLDL) particles are deposited in human atherosclerotic lesions during atherogenesis. Here we studied whether OxLDL-IgG immune complexes (OxLDL-IgG ICs) affect survival of human monocytes. METHODS AND RESULTS: As demonstrated by light microscopy, and analysis of cell proliferation, caspase-3 activity, and DNA fragmentation, OxLDL-IgG ICs promoted survival of cultured human monocytes by decreasing their spontaneous apoptosis. OxLDL-IgG ICs induced a concentration-dependent production of the major monocyte growth factor, monocyte colony-stimulating factor (M-CSF), by the monocytes, but its inhibition was without effect on OxLDL-IgG IC-induced monocyte survival. Rather, OxLDL-IgG ICs induced rapid phosphorylation of Akt, suggesting a direct anti-apoptotic effect mediated by cross-linking of Fcgamma receptors. Experiments with receptor blocking antibodies revealed that the OxLDL-IgG IC-induced monocyte survival was mediated by Fcgamma receptor I. CONCLUSIONS: The results show that OxLDL-IgG ICs promote survival of monocytes by cross-linking Fcgamma receptor I and activating Akt-dependent survival signaling. The results reveal a novel mechanism by which an immune reaction toward oxLDL can play a role in the accumulation of macrophages in human atherosclerotic lesions.

C-reactive protein binds to the 3beta-OH group of cholesterol in LDL particles.

C-reactive protein (CRP) has been suggested to contribute to the development of atherosclerosis. We previously found binding of CRP to cholesterol in modified low density lipoprotein (LDL) particles. Here, we characterize the interaction between CRP and cholesterol in more detail. When lipids of native LDL were separated by thin-layer chromatography, CRP bound only to cholesterol. When various cholesterol analogues were compared for their ability to bind CRP, we found that any modification of the 3beta-OH group blocked binding of CRP to cholesterol. Similarly, enrichment of LDL with cholesterol but not with its analogues triggered the binding of CRP to LDL. Finally, with the aid of anti-CRP monoclonal antibodies and by molecular modeling, we obtained evidence for involvement of the phosphorylcholine-binding site of CRP in cholesterol binding. Thus, CRP can bind to cholesterol, and the interaction is mediated by the phosphorylcholine-binding site of CRP and the 3beta-hydroxyl group of cholesterol.

Effects of acid sphingomyelinase deficiency on male germ cell development and programmed cell death.

Deficiency of acid sphingomyelinase (ASM), an enzyme responsible for producing a pro-apoptotic second messenger ceramide, has previously been shown to promote the survival of fetal mouse oocytes in vivo and to protect oocytes from chemotherapy-induced apoptosis in vitro. Here we investigated the effects of ASM deficiency on testicular germ cell development and on the ability of germ cells to undergo apoptosis. At the age of 20 weeks, ASM knock-out (ASMKO) sperm concentrations were comparable with wild-type (WT) sperm concentrations, whereas sperm motility was seriously affected. ASMKO testes contained significantly elevated levels of sphingomyelin at the age of 8 weeks as detected by high-performance, thin-layer chromatography. Electron microscopy revealed that the testes started to accumulate pathological vesicles in Sertoli cells and in the interstitium at the age of 21 days. Irradiation of WT and ASMKO mice did not elevate intratesticular ceramide levels at 16 h after irradiation. In situ end labeling of apoptotic cells also showed a similar degree of cell death in both groups. After a 21-day recovery period, the numbers of primary spermatocytes and spermatogonia at G2 as well as spermatids were essentially the same in the WT and ASMKO testes, as detected by flow cytometry. In serum-free cultures both ASMKO and WT germ cells showed a significant increase in the level of ceramide, as well as massive apoptosis. In conclusion, ASM is required for maintenance of normal sphingomyelin levels in the testis and for normal sperm motility, but not for testicular ceramide production or for the ability of the germ cells to undergo apoptosis.

Mast cells in neovascularized human coronary plaques store and secrete basic fibroblast growth factor, a potent angiogenic mediator.

OBJECTIVE: Intraplaque neovascularization and hemorrhage may facilitate plaque progression. We studied expression of basic fibroblast growth factor (bFGF), a potent angiogenic mediator, by mast cells (MCs) in human coronary plaques with increasing degrees of atherosclerosis. METHODS AND RESULTS: Normal and atherosclerotic coronary segments were collected from 30 autopsied subjects. Immunohistochemical methods were used to detect MCs, bFGF, and microvessels. Both adventitial and intimal MCs showed intracytoplasmic granular staining for bFGF, and bFGF-positive extracellular granules were observed close to the MCs. Increased numbers of bFGF-positive MCs were detected in neovascularized areas of plaques, and there was a positive correlation between numbers of bFGF-positive MCs and microvessels in both the intima and adventitia. In plaques, the highly neovascularized areas contained increased numbers of bFGF-positive MCs compared with the adjacent nonvascularized areas, where only few MCs were present. Importantly, the proportion of intimal MCs expressing bFGF increased with increasing severity of atherosclerosis. CONCLUSIONS: The present work reveals a novel source of bFGF in human coronary arteries, the intimal and adventitial MCs. The association of bFGF-positive MCs with microvessels and with the severity of atherosclerosis suggests that coronary MCs, by releasing bFGF, may play a role in angiogenesis and progression of coronary plaques.

Cysteine protease cathepsin F is expressed in human atherosclerotic lesions, is secreted by cultured macrophages, and modifies low density lipoprotein particles in vitro.

During atherogenesis, low density lipoprotein (LDL) particles in the arterial intima become modified and fuse to form extracellular lipid droplets. Proteolytic modification of apolipoprotein (apo) B-100 may be one mechanism of droplet formation from LDL. Here we studied whether the newly described acid protease cathepsin F can generate LDL-derived lipid droplets in vitro. Treatment of LDL particles with human recombinant cathepsin F led to extensive degradation of apoB-100, which, as determined by rate zonal flotation, electron microscopy, and NMR spectroscopy, triggered both aggregation and fusion of the LDL particles. Two other acid cysteine proteases, cathepsins S and K, which have been shown to be present in the arterial intima, were also capable of degrading apoB-100, albeit less efficiently. Cathepsin F treatment resulted also in enhanced retention of LDL to human arterial proteoglycans in vitro. Cultured monocyte-derived macrophages were found to secrete active cathepsin F. In addition, similarly with cathepsins S and K, cathepsin F was found to be localized mainly within the macrophage-rich areas of the human coronary atherosclerotic plaques. These results suggest that proteolytic modification of LDL by cathepsin F may be one mechanism leading to the extracellular accumulation of LDL-derived lipid droplets within the proteoglycan-rich extracellular matrix of the arterial intima during atherogenesis.

Protection from radiation-induced male germ cell loss by sphingosine-1-phosphate.

Male germ cells are susceptible to radiation-induced injury, and infertility is a common problem after total-body irradiation. Here we investigated, first, the effects of irradiation on germ cells in mouse testis and, second, the role of sphingosine-1-phosphate (S1P) treatment in radiation-induced male germ cell loss. Irradiation of mouse testes mainly damaged the early developmental stages of spermatogonia. The damage was seen by means of DNA flow cytometry 21 days after irradiation as decreasing numbers of spermatocytes and spermatids with increasing amounts of ionizing radiation (0.1-2.0 Gy). Intratesticular injections of S1P given 1-2 h before irradiation (0.5 Gy) did not protect against short-term germ cell loss as measured by in situ end labeling of DNA fragmentation 16 h after irradiation. However, after 21 days, in the S1P-treated testes, the numbers of primary spermatocytes and spermatogonia at G2 (4C peak as measured by flow cytometry) were higher at all stages of spermatogenesis compared with vehicle-treated testes, indicating protection of early spermatogonia by S1P, whereas the spermatid (1C) populations were similar. In conclusion, S1P appears to protect partially (16%-47%) testicular germ cells against radiation-induced cell death. This warrants further studies aimed at development of therapeutic agents capable of blocking sphingomyelin-induced pathways of germ cell loss.

Sphingosine-1-phosphate in inhibition of male germ cell apoptosis in the human testis.

It has been suggested that apoptosis is controlled by two intracellular sphingolipids, ceramide and sphingosine-1-phosphate (S1P), which are widely distributed in mammalian tissues. In the ovary, S1P was found to effectively block apoptosis caused by cancer therapies. Its role in male germ cell death, however, was unknown. In this study, we investigated the effects of ceramide and S1P on human male germ cell apoptosis. Germ cell death was induced by incubation of segments of seminiferous tubules in vitro. During apoptosis, ceramide levels increased rapidly before appearance of caspase 3 activation and DNA laddering, suggesting a role for ceramide in the induction of germ cell death. Ceramide appeared to regulate an early step of apoptosis because n-acetyl-L-cysteine and blockade of mitochondrial respiration inhibited apoptosis but had no effect on ceramide levels. Moreover, fumonisin B1 (ceramide synthetase inhibitor) did not significantly affect testicular apoptosis. Therefore, elevated ceramide levels are likely to result from breakdown of sphingomyelin rather than from de novo synthesis. Finally, we found that S1P at 1 and 10 micromol/liter suppressed germ cell apoptosis by 30% (P < 0.001). Taken together, sphingolipids appear to play a role in male germ cell apoptosis and can partly be inhibited by S1P.