Increased human tumor necrosis factor‐α levels induce procoagulant change in porcine endothelial cells in vitro

Lee, Kyoung Geun; Lee, Haneulnari; Ha, Joo Young; Lee, Young Kyung; Kang, Hee Jung; Park, Chung Gyu; Kim, Sang Joon

doi:10.1111/j.1399-3089.2012.00704.x

Cited by 14 publications

(11 citation statements)

References 38 publications

(53 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Coagulation time was measured as described by Lee et al. . To measure coagulation time induced by human plasma against porcine endothelial cells, experimental cells were suspended as single cells using 0.25% EDTA‐trypsin (Invitrogen) and each group (4 × 10 4 ) in 50 μ l EBM (Clonetics) was then mixed with the same volume of normal citrated platelet‐poor plasma.…”

Section: Methodsmentioning

confidence: 99%

Human thrombomodulin regulates complement activation as well as the coagulation cascade in xeno‐immune response

Kim

Hawthorne

Kang

et al. 2015

Xenotransplantation

Self Cite

View full text Add to dashboard Cite

Background: With the introduction of the a1, 3-galactosyltransferase gene-knockout (GT-KO) pig and its pivotal role in preventing hyperacute rejection (HAR), coagulation remains a considerable obstacle yet to be overcome in order to provide long-term xenograft survival. Thrombomodulin (TBM) plays a critical anticoagulant and anti-inflammatory role in its part of the protein C pathway. Many studies have demonstrated the strong anticoagulant effects of TBM in xenotransplantation, but its complement regulatory effects have not been appropriately examined. Here, we investigate whether TBM can regulate complement activation as well as coagulation in response to xenogeneic stimuli. Methods: We transfected porcine endothelial cells (MPN-3) with adenovirus vectors containing the human TBM gene (ad-hTBM), or a control gene containing GFP (ad-GFP). The expression level of ad-hTBM was measured by flow cytometry. To confirm the anticoagulant effect of TBM, coagulation time was measured after treatment with recalcified human plasma in adhTBM-transfected MPN-3, and a thrombin activity assay was performed after treatment with 50% human serum in ad-hTBM-infected MPN-3. Results: Thrombin generation was significantly decreased in a dosedependent manner in ad-TBM group, and coagulation time was increased in the ad-hTBM group when compared to the ad-GFP group. Complement-dependent serum toxicity assays were performed after treatment with 20% human serum or heat-inactivated human serum by LDH assay. Complement-dependent toxicity was significantly attenuated in the ad-hTBM group, but complement-independent toxicity was not attenuated in the ad-hTBM group. These results suggest that human thrombomodulin (hTBM) has complement regulatory effects as well as anticoagulant effects. To further investigate the mechanisms of complement regulation by hTBM, we deleted the EGF5, 6 domains that are involved in thrombin generation or the lectin-like domain involved in inflammation of TBM and functional tests were performed using these modified forms. We showed that the EGF5, 6 domain of TBM principally inhibits complement activation rather than the lectin domain. Conclusion: The EGF5, 6 domains of TBM appear to be the major domains for down-regulating the complement system rather than the lectin-like domain during xenogenic stimuli. The role of EGF5, 6 domains of hTBM may be due to inhibition of thrombin as thrombin can cleave C3a and C5a directly and hTBM may also be involved in complement regulation. Clearly then human TBM has complement regulatory effects as well as anticoagulant effects in xeno-immune response, and it is a promising target for attenuating xenograft rejection.

show abstract

Section: Methodsmentioning

confidence: 99%

Human thrombomodulin regulates complement activation as well as the coagulation cascade in xeno‐immune response

Kim

Hawthorne

Kang

et al. 2015

Xenotransplantation

Self Cite

View full text Add to dashboard Cite

show abstract

“…Several studies have demonstrated that both FM and LM contribute to the determination of bone mass [ 7 , 8 ] while others have found that LM has a greater impact on BMD and may protect against the risk of fractures [ 9 – 12 ]. On the other hand, other studies have suggested that FM, rather than LM, is the most important determining factor for BMD [ 13 – 15 ] and that FM might positively contribute to BMD only in older males [ 16 ]. The discrepant findings of these studies suggest that BMD is dependent on a variety of factors including gender, ethnicity, BMI, and age.…”

Section: Introductionmentioning

confidence: 99%

Aged-Related Changes in Body Composition and Association between Body Composition with Bone Mass Density by Body Mass Index in Chinese Han Men over 50-year-old

et al. 2015

View full text Add to dashboard Cite

ObjectivesAging, body composition, and body mass index (BMI) are important factors in bone mineral density (BMD). Although several studies have investigated the various parameters and factors that differentially influence BMD, the results have been inconsistent. Thus, the primary goal of the present study was to further characterize the relationships of aging, body composition parameters, and BMI with BMD in Chinese Han males older than 50 years.MethodsThe present study was a retrospective analysis of the body composition, BMI, and BMD of 358 Chinese male outpatients between 50 and 89 years of age that were recruited from our hospital between 2009 and 2011. Qualified subjects were stratified according to age and BMI as follows: 50–59 (n = 35), 60–69 (n = 123), 70–79 (n = 93), and 80–89 (n = 107) years of age and low weight (BMI: < 20 kg/m2; n = 21), medium weight (20 ≤ BMI < 24 kg/m2; n = 118), overweight (24 ≤ BMI < 28 kg/m2; n = 178), and obese (BMI ≥ 28 kg/m2; n = 41). Dual-energy X-ray absorptiometry (DEXA) was used to assess bone mineral content (BMC), lean mass (LM), fat mass (FM), fat-free mass (FFM), lumbar spine (L1-L4) BMD, femoral neck BMD, and total hip BMD. Additionally, the FM index (FMI; FM/height2), LM index (LMI; LM/height2), FFM index (FFMI; [BMC+LM]/height2), percentage of BMC (%BMC; BMC/[BMC+FM+LM] × 100%), percentage of FM (%FM; FM/[BMC+FM+LM] × 100%), and percentage of LM (%LM; LM/(BMC+FM+LM) × 100%) were calculated. Osteopenia or osteoporosis was identified using the criteria and T-score of the World Health Organization.ResultsAlthough there were no significant differences in BMI among the age groups, there was a significant decline in height and weight according to age (p < 0.0001 and p = 0.0002, respectively). The LMI and FFMI also declined with age (both p < 0.0001) whereas the FMI exhibited a significant increase that peaked in the 80-89-years group (p = 0.0145). Although the absolute values of BMC and LM declined with age (p = 0.0031 and p < 0.0001, respectively), there was no significant difference in FM. In terms of body composition, there were no significant differences in %BMC but there was an increase in %FM (p < 0.0001) and a decrease in %LM (p < 0.0001) with age. The femoral neck and total hip BMD significantly declined with age (p < 0.0001 and p = 0.0027, respectively) but there were no differences in L1-L4. BMD increased at all sites (all p < 0.01) as BMI increased but there were declines in the detection rates of osteoporosis and osteopenia (both p < 0.001). A logistic regression revealed that when the medium weight group was given a BMI value of 1, a decline in BMI was an independent risk factor of osteoporosis or osteopenia, while an increase in BMI was a protective factor for BMD. At the same time, BMD in L1-L4 exhibited a significant positive association with FMI (p = 0.0003) and the femoral neck and total hip BMDs had significant positive associations with FFMI and LMI, respectively (both p < 0.0001).ConclusionsThese data indicate that LMI and FFMI exhibited significant ...

show abstract

“…As the initiator of the extrinsic coagulation cascade, TF plays the key role in dysregulation of vascular homeostasis. It was shown that TNF‐α levels increased in human blood after contact with porcine endothelial cells, which in turn led to procoagulant changes and upregulation of TF . Even further genetic modifications targeting complement activation and the immune system would not overcome these changes since it was reported that human monocytes can be activated by PAEC to express TF independently of natural antibodies and complement .…”

Section: Discussionmentioning

confidence: 99%

siRNA Mediated Knockdown of Tissue Factor Expression in Pigs for Xenotransplantation

Ahrens

Petersen

Herrmann

et al. 2015

American Journal of Transplantation

View full text Add to dashboard Cite

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 AE 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-a was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 AE 136 vs. WT: 13 038 AE 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 AE 8.8 min, WT: 45.8 AE 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 AE 13.0, TFkd: 2.6 AE 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multitransgenic pigs for xenotransplantation.

show abstract

Increased human tumor necrosis factor‐α levels induce procoagulant change in porcine endothelial cells in vitro

Cited by 14 publications

References 38 publications

Human thrombomodulin regulates complement activation as well as the coagulation cascade in xeno‐immune response

Human thrombomodulin regulates complement activation as well as the coagulation cascade in xeno‐immune response

Aged-Related Changes in Body Composition and Association between Body Composition with Bone Mass Density by Body Mass Index in Chinese Han Men over 50-year-old

siRNA Mediated Knockdown of Tissue Factor Expression in Pigs for Xenotransplantation

Contact Info

Product

Resources

About