Effects of HSP70.1/3 gene knockout on acute respiratory distress syndrome and the inflammatory response following sepsis

2006 ◽  
Vol 290 (5) ◽  
pp. L956-L961 ◽  
Author(s):  
Kristen D. Singleton ◽  
Paul E. Wischmeyer
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Lung Tissue ◽  
Inflammatory Cytokine ◽  
Gene Knockout ◽  
Cecal Ligation ◽  
Hsp70 Expression ◽  
Experimental Sepsis ◽  
Hsp70 Gene ◽  
Tnf Α

Heat shock response has been implicated in attenuating NF-κB activation and inflammation following sepsis. Studies utilizing sublethal heat stress or chemical enhancers to induce in vivo HSP70 expression have demonstrated survival benefit after experimental sepsis. However, it is likely these methods of manipulating HSP70 expression have effects on other stress proteins. The aim of this study was to evaluate the role of specific deletion of HSP70.1/3 gene expression on ARDS, NF-κB activation, inflammatory cytokine expression, and survival following sepsis. To address this question, we induced sepsis in HSP70.1/3 KO and HSP70.1/3 WT mice via cecal ligation and puncture (CLP). We evaluated lung tissue NF-κB activation and TNF-α protein expression at 1 and 2 h, IL-6 protein expression at 1, 2, and 6, and lung histopathology 24 h after sepsis initiation. Survival was assessed for 5 days post-CLP. NF-κB activation in lung tissue was increased in HSP70.1/3(−/−) mice at all time points after sepsis initiation. Deletion of HSP70.1/3 prolonged NF-κB binding/activation in lung tissue. Peak expression of lung TNF-α at 1 and 2 h was also significantly increased in HSP70.1/3(−/−) mice. Expression of IL-6 was significantly increased at 2 and 6 h, and histopathology revealed a significant increase in lung injury in HSP70.1/3(−/−) mice. Last, deletion of the HSP70 gene led to increased mortality 5 days after sepsis initiation. These data reveal that absence of HSP70 alone can significantly increase ARDS, activation of NF-κB, and inflammatory cytokine response. The specific absence of HSP70 gene expression also leads to increased mortality after septic insult.

Mechanisms of MAdCAM-1 gene expression in human intestinal microvascular endothelial cells

2005 ◽  
Vol 288 (2) ◽  
pp. C272-C281 ◽  
Author(s):  
Hitoshi Ogawa ◽  
David G. Binion ◽  
Jan Heidemann ◽  
Monica Theriot ◽  
Pamela J. Fisher ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Protein Expression ◽  
Molecular Mechanisms ◽  
Primary Cultures ◽  
Microvascular Endothelial Cells ◽  
Cellular Density ◽  
Tnf Α ◽  
Gene And Protein Expression ◽  
Microvascular Endothelial

Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is a homing receptor preferentially expressed on gut-associated endothelial cells that plays a central role in leukocyte traffic into the mucosal immune compartment. Although the molecular mechanisms underlying endothelial ICAM-1 or E-selectin expression have been intensively investigated, the mechanisms that regulate human MAdCAM-1 expression have not been defined. We report MAdCAM-1 gene and protein expression in primary cultures of human intestinal microvascular endothelial cells (HIMEC) that was not demonstrated in human umbilical vein endothelial cells. Similar to ICAM-1 and E-selectin expression, MAdCAM-1 gene expression in HIMEC was inducible with TNF-α, IL-1β, or LPS activation. However, in striking contrast to ICAM-1 and E-selectin expression, MAdCAM-1 mRNA and protein expression in HIMEC was heavily dependent on culture duration and/or cellular density, suggesting a prominent role for cell-cell interaction among these endothelial cells in the expression of the mucosal addressin. MAdCAM-1 expression was inhibited by both SN-50 (NF-κB inhibitor) and LY-294002 [phosphatidylinositol 3-kinase (PI3-K) inhibitor], whereas ICAM-1 and E-selectin expression was inhibited by SN-50 but not by LY-294002. The Akt phosphorylation by TNF-α or LPS was greater at higher cell density, demonstrating a pattern similar to that of MAdCAM-1 expression. NF-κB activation was not affected by cellular density in HIMEC. MAdCAM-1 expression in human gut endothelial cells is regulated by distinct signaling mechanisms involving both NF-κB and PI3-K/Akt. These data also suggest that PI3-K/Akt is involved in the gut-specific differentiation of HIMEC, which results in expression of the mucosal addressin MAdCAM-1.

scholarly journals Lactobacillus casei Strain Shirota Enhances the Ability of Geniposide to Activate SIRT1 and Decrease Inflammation and Oxidative Stress in Septic Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Chao Mai ◽  
Li Qiu ◽  
Yong Zeng ◽  
Xingqin Tan
Keyword(s):  
Oxidative Stress ◽  
Protein Expression ◽  
Lactobacillus Casei ◽  
P53 Protein ◽  
Cecal Ligation ◽  
Morris Water Maze Test ◽  
Gardenia Jasminoides ◽  
Tnf Α ◽  
Anti Oxidant ◽  
And Oxidative Stress

Gardenia jasminoides Ellis is rich in geniposide, which can be transformed into the anti-oxidant and anti-inflammatory agent genipin. Genipin exhibits greater efficacy than geniposide, but it is unstable and difficult to preserve. In this study, a mouse model for sepsis was established by cecal ligation and puncture, and then we explored the effects and mechanism of Lactobacillus casei strain Shirota (LcS) on the enhancement of the ability of geniposide to reduce sepsis and decrease inflammatory and oxidative levels in mice by the regulation of sirtuin type 1 (SIRT1). The mice were evaluated and analyzed by the open field test, Morris water maze test, flow cytometry, kit assay, qPCR, and western blot. The LcS + geniposide increased the survival rate in mice with sepsis, and increased the total travel distance, number of times the mice stood up, amount of time the mice spent grooming their fur, duration in the target quadrant, and crossing area number. The testing of mouse nerve cells showed that LcS + geniposide reduced the rate of nerve cell apoptosis caused by sepsis. LcS + geniposide also decreased the amount of inflammatory-related indicators of TNF-α, IL-6, and IL-1β, and the oxidation-related levels of malondialdehyde (MDA) in the hippocampi of septic mice, and it increased the oxidase activities of superoxide dismutase (SOD) and catalase (CAT). Additionally, LcS + geniposide increased the SOD1, SOD2, and CAT mRNA expression in the hippocampi of mice with sepsis and decreased the expression of TNF-α, IL-1β, NF-κB, and p53 mRNA. LcS+geniposide also increased the SIRT1 protein expression and decreased the Ac-FOXO1, Ac-NF-κB, and Ac-p53 protein expression in the hippocampi of mice with sepsis. We also observed that LcS + geniposide decreased the inflammatory and oxidative damage in the mice with sepsis. The effect of LcS + geniposide was similar to that of the drug dexamethasone and stronger than the effect of geniposide utilized alone. LcS also enhanced the ability of geniposide to activate SIRT1 and decrease the inflammation and oxidative stress in the septic mice, and it achieved an effect same with that obtained by the use of the drug dexamethasone.

scholarly journals Dysregulated Macrophage Pro-Inflammatory Cytokine Production and Chronic Colitis in Mice Lacking Both Gab2 and Gab3

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 457-457
Author(s):  
Tamisha Y. Vaughan-Whitley ◽  
Hikaru Nishio ◽  
Barry Imhoff ◽  
Zhengqi Wang ◽  
Silvia T. Bunting ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Protein Expression ◽  
Knockout Mice ◽  
Inflammatory Cytokine ◽  
Cytokine Production ◽  
Wild Type ◽  
Inflammatory Cytokine Production ◽  
Tnf Α ◽  
Single Knockout

Abstract Macrophages are responsible for protecting the body against foreign invaders. We have been studying the role of Grb2-associated binding proteins (Gabs) in macrophage biology. In mice, Gabs are adaptor proteins that include three family members (Gab1, Gab2, and Gab3) that play critical regulatory roles in modulating cytokine receptor signaling. Gab2 knockout mice have no developmental defects but have impaired allergic responses, osteoclast defects, altered mast cell development, and altered hematopoiesis. Gab3 knockout mice have no defined phenotypes alone and although highly expressed in macrophages, a functional role was not found despite considerable focus on this cell type. Therefore, we set out to determine the combined role of Gab2 and Gab3 to determine whether they performed redundant functions not observable in single knockout mice. To analyze regulation of macrophage cytokine production, a Gab2/3 deficient mouse model was generated on the C57BL/6 background. Bone Marrow Derived Macrophages (BMDM) were expanded from the bone marrow (BM) of wild-type (WT), Gab2 and Gab3 single knockout and Gab2/3 knockout mice and found to similarly co-express CD11b and F4/80. However, Gab2/3 knockout BM produced only 30% of wild-type BMDM numbers. Despite reductions in BMDM absolute numbers, isolated BMDM demonstrated significant induction of pro-inflammatory cytokines TNF-α and IL-12 and anti-inflammatory cytokine IL-10 mRNA at baseline. Interestingly, after LPS stimulation (100ng/ml) we detected much greater induction of TNF-α and IL-12 mRNA and protein expression. Interestingly, despite increased IL-10 mRNA induction in Gab2/3 knockout BMDM, no IL-10 protein expression could be detected by Luminex assay. No changes were observed in production of interferon or STAT1 activation in these BMDM. Studies have shown that rapamycin treatment of macrophages suppresses mTORC1 and subsequently reduces IL-10 production and promotes pro-inflammatory cytokine production. Gab2 is known for its role in regulating the PI3K pathway through interactions with the p85 regulatory subunit of PI3K. Therefore, we also examined whether mTOR activation was effected by Gab2/3 deficiency causing altered cytokine expression. Deletion of Gab2/3 in BMDMs treated with LPS showed an inhibition of 4EBP1 phosphorylation and increased AKT phosphorylation. These results suggest that Gabs may play a critical role in modulating mTOR activation and potentially causing defects in protein translation that reflect in reduced IL-10 cytokine levels in Gab2/3 knockout cells. IL-10 has a critical immunoregulatory role that is dysregulated in patients with inflammatory bowel disease. IL-10 deficient mice develop colitis due to loss of mucosal immune tolerance. Strikingly, as early as two months of age in vivo 12/32 (37.5%) Gab2/3 knockout mice developed rectal prolapse and suffered from diarrhea within a six month period. Histological analysis of isolated colons using a scoring system confirmed spontaneous development of colitis in Gab2/3 knockout mice compared to no phenotypes observed in WT and single knockout controls. To determine whether the BM was directly involved in the disease, BM chimeras were generated using irradiated WT mice as recipients and Gab2/3 knockout mice as donors. Susceptible recipients receiving Gab2/3 knockout BM showed a more invasive colitis phenotype than the spontaneous disease and resulted in forced euthanization due to body weight decreases greater than 25%. Multiple ulcerations were present in most of the colon proximal region, with extensive epithelial damage, transmural inflammation, and in some mice adenocarcinoma. Notably, we did not observe adenocarcinoma in untransplanted Gab2/3 knockout mice, suggesting that epithelial deletion of Gab2/3 may suppress cancer whereas in the bone marrow chimera model, the epithelial cells are WT and can be transformed. Similar phenotypes were also observed in secondary transplant recipients. Lastly, treatment of Gab2/3 knockout mice with dextran-sodium-sulfate (DSS) induced rapid severe colitis that resulted in death of 80% and 40% of Gab2/3 knockout and WT mice respectively. Overall, these observations demonstrate a major redundant role for Gab2 and Gab3 in macrophage immune surveillance required for the prevention of colitis in mice. Disclosures No relevant conflicts of interest to declare.

scholarly journals miR-20a attenuates acute lung injury in septic rats via targeting TLR4

2021 ◽  
Keyword(s):  
Acute Lung Injury ◽  
Lung Injury ◽  
Inflammatory Response ◽  
Lung Tissue ◽  
Cecal Ligation ◽  
Normal Group ◽  
Lung Damage ◽  
Luciferase Reporter ◽  
Arterial Bleeding ◽  
Tnf Α

Background: Sepsis is most likely to cause lung damage in patients, and the detection rate and mortality rate are high. Here, we investigated the expression of miR-20a in sepsis-induced acute lung injury (ALI) rats and its effect on inflammatory response, and reveal its possible molecular mechanism. Method: The model of acute lung injury caused by sepsis in rats was established by cecal ligation and puncture. The expression of miR-20a in lung tissue was determined by RT-qPCR. Acute lung injury rats were injected with 5 nmol miR-20a agomir or agomir NC every day for 3 days. Rats were sacrificed by arterial bleeding and lung tissues were removed. Serum interleukin (IL) -1β, IL-6, and tumor necrosis factor alpha (TNF-α) were detected by ELISA. HE staining was used to observe the pathology of lung tissue and calculate the pathological score of lung injury. Western blot to determine the level of TLR4 and nuclear transcription factor κB p65 (NF-κB p65) protein in lung tissue. The luciferase reporter assay was used to verify the binding effect of miR-20a on the 3 non-coding TLR4. Results: We found that compared with that in Normal group, the expression of miR-20a in lung tissues of rats with ALI was decreased (p < 0.05). In miR-20a agomir group, the plasma level of IL-1β, IL-6, and TNF-α was significantly lower than that in agomir NC group and ALI group (p < 0.05), while higher than those in Normal group (p < 0.05). The HE staining results showed that the pathological score of lung injury in rats in miR-20a agomir group was lower than that of agomir NC group and ALI group (p < 0.05). Compared with agomir NC group and ALI group, the expression of TLR4 and NF-κB p65 in miR-20a agomir group was decreased (p < 0.01). The luciferase reporting experiment confirmed that TLR4 was a target gene of miR-20a. Conclusion: To sum up, miR-20a exerts a protective effect on sepsis-induced ALI rats through its anti-inflammatory effect. The targeting of TLR4 by miR-20a may be an effective method to reduce the inflammatory response in sepsis-induced ALI.

scholarly journals Regulation of dendritic cell function improves survival in experimental sepsis through immune chaperone

2019 ◽  
Vol 25 (4) ◽  
pp. 235-243 ◽  
Author(s):  
Pengfei Li ◽  
Ran Zhao ◽  
Kevin Fan ◽  
Stephen Iwanowycz ◽  
Hongkuan Fan ◽  
...  
Keyword(s):  
Cell Function ◽  
Critical Role ◽  
Cecal Ligation ◽  
Experimental Sepsis ◽  
Adaptive Immune ◽  
Sepsis Model ◽  
Novel Approach ◽  
Tnf Α ◽  
Heat Shock Protein Gp96 ◽  
Dc Maturation

Dendritic cells (DCs) are professional Ag-presenting cells that play a critical role in both innate and adaptive immune responses. DCs recognize and respond to bacteria through multiple PRRs, including TLRs. Heat shock protein gp96/grp94 is a master essential chaperone for TLRs in the endoplasmic reticulum. We generated DC-specific gp96-knockout (KO) mice and showed that gp96 KO DCs were unable to respond to multiple TLR ligands. TLR-mediated hyperinflammatory response can lead to sepsis. However, the roles of neither DCs nor the DC-intrinsic gp96 in the process are completely understood. In a LPS-induced sepsis model, we hereby found that deletion of gp96 in DCs significantly reduced serum TNF-α levels and improved survival. Furthermore, using the well-defined polymicrobial sepsis model of cecal ligation and puncture, we found that DC-specific ablation of gp96 improved survival with significantly attenuated liver and renal injuries, decreased circulating inflammatory cytokines, altered DC maturation and activation, and increased serum Ig. Collectively, we demonstrate that deletion of gp96 in DCs is beneficial in protecting mice against sepsis induced by both endotoxemia and polymicrobial infections. We conclude that targeting gp96 in DCs may provide a potential novel approach for reducing the morbidity and mortality of sepsis.

scholarly journals Angiotensin II AT1 blockade reduces the lipopolysaccharide-induced innate immune response in rat spleen

2009 ◽  
Vol 296 (5) ◽  
pp. R1376-R1384 ◽  
Author(s):  
Enrique Sánchez-Lemus ◽  
Julius Benicky ◽  
Jaroslav Pavel ◽  
Ignacio M. Larrayoz ◽  
Jin Zhou ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
Inflammatory Response ◽  
Protein Expression ◽  
Innate Immune Response ◽  
Innate Immune ◽  
Bacterial Endotoxin ◽  
Ang Ii ◽  
Tnf Α ◽  
Mrna And Protein Expression

ANG II AT1 receptor blockade reduces inflammation in hypertension. To determine whether ANG II AT1 receptor blockers (ARBs) influence the innate immune inflammatory response in normotensive rats, we studied rat plasma and spleen after a 3-day subcutaneous pretreatment with the ARB candesartan followed by a single dose of the bacterial endotoxin LPS (50 μg/kg ip). Peripheral administration of LPS to rodents produced a generalized inflammatory response with increased release of TNF-α, IL-1β, and IL-6 into the circulation. Candesartan pretreatment reduced the LPS-induced release of TNF-α, IL-1β, and IL-6 into the circulation. The red pulp of rat spleen expressed large numbers of AT1 receptors and the LPS receptors Toll-like receptor 4 and CD14. Candesartan administration significantly blocked AT1 receptors. The ARB reduced the LPS-induced upregulation of CD14 gene expression; expression of TNF-α and IL-6 mRNA and protein; expression of IL-1β and IκB-α mRNA; COX-2 mRNA and protein expression and PGE2 concentration; inducible nitric oxide synthase (iNOS) gene and protein expression and iNOS activity; and Nox2 gene expression and 8-isoprostane levels. In addition, candesartan reduced the CD14 protein expression in saline- and LPS-treated rats. Our results suggest that AT1 receptors are essential for the development of the full innate immune response to bacterial endotoxin. The ARB decreased the general peripheral inflammatory reaction to LPS and partially decreased the inflammatory response in the spleen. An unrestricted innate immune response to the bacterial endotoxin may have deleterious effects for the organism and may lead to development of chronic inflammatory disease. We postulate that ARBs may have therapeutic effects on inflammatory conditions.

Endotoxin downregulates peroxisome proliferator-activated receptor-γ via the increase in TNF-α release

2008 ◽  
Vol 294 (1) ◽  
pp. R84-R92 ◽  
Author(s):  
Mian Zhou ◽  
Rongqian Wu ◽  
Weifeng Dong ◽  
Asha Jacob ◽  
Ping Wang
Keyword(s):  
Gene Expression ◽  
Neutralizing Antibodies ◽  
Cecal Ligation ◽  
Polymyxin B ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Protein Levels ◽  
Ppar Γ ◽  
Tnf Α ◽  
A Cell

The nuclear receptor peroxisome proliferator-activated receptor-γ (PPAR-γ) is anti-inflammatory in a cell-based system and in animal models of endotoxemia. We have shown that PPAR-γ gene expression is downregulated in macrophages after lipopolysaccharide (LPS) stimulation. However, it remains unknown whether hepatic PPAR-γ is altered in sepsis and, if so, whether LPS directly downregulates PPAR-γ. To study this, rats were subjected to sepsis by cecal ligation and puncture (CLP). Hepatic tissues were harvested at 5, 10, and 20 h after CLP. PPAR-γ gene expression and protein levels were determined by RT-PCR and Western blot analysis, respectively. The results showed that PPAR-γ gene expression decreased at 10 and 20 h and that its proteins levels were reduced at 20 h after CLP. PPAR-γ levels were also decreased in animals that were administered LPS. To determine the direct effects of LPS on PPAR-γ downregulation, LPS binding agent polymyxin B (PMB) was administered intramuscularly after CLP. The administration of PMB significantly reduced plasma levels of endotoxin, but it did not prevent the downregulation of PPAR-γ expression. We found that circulating levels of TNF-α still remained significantly elevated in PMB-treated septic animals. We, therefore, hypothesize that the decrease of PPAR-γ expression is TNF-α dependent. To investigate this, Kupffer cells (KCs) were isolated from normal rats and stimulated with LPS or TNF-α. TNF-α significantly attenuated PPAR-γ gene expression in KCs. Although LPS decreased PPAR-γ in KCs, the downregulatory effect of LPS was blocked by the addition of TNF-α-neutralizing antibodies. Furthermore, the administration of TNF-α-neutralizing antibodies to animals before the onset of sepsis prevented the downregulation of PPAR-γ in sepsis. We, therefore, conclude that LPS downregulates PPAR-γ expression during sepsis via an increase in TNF-α release.

scholarly journals Effect of betaine on HSP70 expression and cell survival during adaptation to osmotic stress

1993 ◽  
Vol 293 (2) ◽  
pp. 553-558 ◽  
Author(s):  
P G Petronini ◽  
E M De Angelis ◽  
A F Borghetti ◽  
K P Wheeler
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Osmotic Stress ◽  
Cell Survival ◽  
Osmotic Pressure ◽  
Colony Formation ◽  
Hsp70 Expression ◽  
Hsp70 Gene ◽  
3T3 Cells ◽  
Induced Expression

Induced expression of the HSP70 gene in 3T3 and SV-3T3 cells was monitored by measurements of the synthesis of HSP70 and of the cellular contents of both HSP70 and its mRNA. The presence of betaine (N-trimethylglycine) at concentrations of 2.5-25 mM decreased the induction of HSP70 gene expression caused by incubation of 3T3 and SV-3T3 cells in hypertonic (0.5 osM) medium. This effect was accompanied by an enhancement of SV-3T3 cell adaptation, assayed by colony formation, to the hyperosmotic conditions. In contrast, the presence of betaine did not affect HSP70 gene expression induced in these cells by heat shock. After 6 h incubation with 25 mM betaine under hypertonic (0.5 osM) conditions the intracellular concentration of betaine in SV-3T3 cells was about 195 mM, compared with about 70 mM under isotonic (0.3 osM) conditions. Hence, with this concentration of extracellular betaine, the marked increase in the accumulation of betaine within the cells presumably counteracts the imposed osmotic pressure and eliminates the signal that otherwise initiates increased expression of the HSP70 gene.

Bortezomib Improves Endothelial Thromboresistance Via Induction of KLF Transcription Factors.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1890-1890
Author(s):  
Toyoko Hiroi ◽  
Clayton B Deming ◽  
Haige Zhao ◽  
Baranda S Hansen ◽  
Elisabeth K Arkenbout ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Protein Expression ◽  
Tissue Factor ◽  
Lung Tissue ◽  
Protein C ◽  
Umbilical Vein ◽  
Small Interfering Rnas ◽  
Gene And Protein Expression

Abstract Background: Patients with multiple myeloma (MM) are at high risk for venothromboembolic events (VTE). Recent studies, however, suggest that MM patients treated with bortezomib, an approved proteasome inhibitor with potent NF-kB inhibitory effects, appear to have a lower risk of VTE compared to those treated with other therapies. We hypothesize that this could be due to a beneficial effect of bortezomib on endothelial thromboresistance. Methods and Results: Human umbilical vein endothelial cells (HUVECs) were incubated with bortezomib for 20 hours and changes in the expression of a panel of coagulation and inflammation-related genes measured by qPCR. Bortezomib stimulated baseline expression of anticoagulant genes (thrombomodulin (TM), eNOS and tissue factor pathway inhibitor), suppressed baseline expression of pro-coagulant genes (vWF and protease activated receptor-1) and suppressed cytokine-mediated induction of E-selectin, VCAM-1 and tissue factor. Most pronounced, was the dose-dependent upregulation of TM, a member of the protein C anticoagulant pathway (229 ± 15% and 341 ± 7% of control, at 5 nM and 10 nM bortezomib, respectively, p &lt;0.0001). Induction of TM gene expression was paralleled by a significant upregulation of TM protein expression, assessed by Western blot analysis, and by an increased capacity to generate activated protein C (205 ± 5% of control with 5 nM bortezomib, p &lt;0.0001). Bortezomib-induced TM upregulation was blocked by cycloheximide, suggesting that induction of a transcriptional pathway, and not simply inhibition of the NF-kB pathway, was required. We therefore examined the effects of bortezomib on the expression of several Krüppel like transcription factors (KLFs) that are known to be important regulators of TM expression and endothelial thromboresistance. Bortezomib significantly upregulated the expression of KLF2, KLF4 and KLF6 in HUVECs (18 ± 1, 8 ± 1 and 2 ± 0.1-fold of control, respectively, p &lt;0.0001 for each) following a 5 nM exposure for 20 hours. Knock-down experiments using small interfering RNAs revealed that KLF2 and KLF4, but not KLF6, play critical and synergistic roles in mediating bortezomib-induced TM upregulation. To determine the in vivo significance of these findings, mice were administered ascending doses of bortezomib for 7 days and TM expression measured in selected tissue. Compared to controls, a dose of 0.8 mg/kg bortezomib ip increased TM gene and protein expression in the liver by 7.0 ± 1.1 and 9.5 ± 2.9-fold, respectively (p &lt;0.0001 for each) and TM gene expression in the kidney by 2.5 ± 0.2-fold (p &lt;0.0001). There was no significant change in TM expression observed in heart and lung tissue. Paralleling changes in TM expression, expression of the KLF2 and KLF4 genes was also increased in the liver (2.1 ± 0.3 and 6.2 ± 1.5-fold of controls, respectively, p &lt;0.001 for each) and kidney (1.9 ± 0.2 and 2.9 ± 0.3-fold of controls, respectively, p &lt;0.01 for each), but not in heart or lung tissue. Conclusions: Bortezomib markedly stimulates endothelial TM expression, both in vitro and in vivo in a tissue-specific manner. TM upregulation appears dependent on the induction of KLF2 and KLF4 transcription factors rather than by inhibition of the NFkB pathways. Our findings provide a rationale for further studies of bortezomib-induced enhancement of endothelial thromboresistance in patients with MM and may help explain why these patients are reduced risk for VTE.

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