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Although the role of nuclear factor kappa B (NF-κB) in the pathogenesis of sepsis and septic shock has been extensively studied, little is known about the causative contribution of endothelial intrinsic NF-κB to these pathological processes. Here, we used transgenic mice (TG on FVB genetic background) that conditionally overexpress the NF-κB inhibitor, I-κBαmt, selectively on endothelium and their transgene negative littermates (WT) to define the causative role of endothelial specific NF-κB signaling in septic shock and septic vascular dysfunction. In WT mice, LPS challenge caused systemic hypotension, a significantly blunted vasoconstrictor response to norepinephrine and an impaired endothelium-dependent vasodilator response to acetylcholine, concomitant with a markedly increased aortic iNOS expression, significantly elevated plasma and aortic levels of nitrite/nitrate, increased aortic TNF-α expression and decreased aortic eNOS expression. In TG mice whose endothelial NF-κB was selectively blocked, LPS caused significantly less hypotension and no impairments in vasoconstrictor and endothelium-dependent vasodilator responses, associated with significantly reduced aortic iNOS expression, decreased plasma and aortic levels of nitrite/nitrate, reduced aortic TNF-α expression and increased aortic eNOS expression. TNF-α knockout mice prevented LPS-induced eNOS down-regulation. WT mice subjected to cecal ligation and puncture showed significant systemic hypotension, which was prevented in TG mice. Our dada show that selective blockade of endothelial intrinsic NF-κB pathway is sufficient to abrogate the cascades of molecular events that lead to septic shock and septic vascular dysfunction, demonstrating a pivotal role of endothelial specific NF-κB signaling in the pathogenesis of septic shock and septic vascular dysfunction.
Septic shock and septic vascular dysfunction are characterized by systemic hypotension, persistent vasodilatation, hyporesponsiveness to vasoconstrictors and impairment of endothelium-dependent vasodilator response. The mechanisms of septic shock and septic vascular dysfunction are complex and multiple (1–3). One well-established mechanism is the activation of NF-κB pathway. Bacterial pathogens and their products, such as LPS, activate NF-κB, which causes inducible nitric oxide (NO) synthase (iNOS) expression (2, 3), leading to an excessive production of NO. NO released subsequently causes vasodilatation, vascular hyporeactivity and hypotension by activating soluble guanylyl cyclase-dependent mechanism (4–7). NF-κB activation mediates the expression of numerous cytokines, which lead to further activation of NF-κB, amplifying and perpetuating the inflammatory response (2). LPS and cytokines, such as TNF-α, down-regulate endothelial NO synthase (eNOS) expression (8, 9), which is believed to be an important molecular event underlying the impaired endothelium-dependent vasodilator response (10, 11). Animal studies have demonstrated that inhibition of NF-κB activation inhibits multiple inflammatory gene expression (2, 12), reverses systemic hypotension (2, 13–15), corrects myocardial dysfunction (16) and prevents the impairment of endothelium-dependent vasodilatation (2, 14).
The pathogenic role of NF-κB activation in septic shock and septic vascular dysfunction is unquestionable. However, the contribution of individual cell specific NF-κB to these pathological processes is significantly less clear. The pathophysiology of sepsis and septic shock involves complex cell-cell and mediator-mediator interactions (1, 2). Emerging evidence suggests that different cell intrinsic NF-κB may play distinct role in the pathophysiology of sepsis (17–19). Elucidation of the contribution of individual cell specific NF-κB to the complicated pathological process of septic shock will help to better understand the pathologic mechanisms of septic shock.
The vascular endothelium is considered an important mechanism regulating vascular tone and vascular homeostasis (4, 20). In response to physiological stimuli and hemodynamic forces, endothelial cells release contracting and relaxing factors including endothelin, NO and prostacyclin. These factors alter vascular tone by directly causing vasoconstriction or vasodilatation, and by synergistically or counteractively interacting with neurotransmitters, enhancing or diminishing the neurally-induced vascular contraction or relaxation (4, 20). Endothelium-derived NO inhibits adrenergic neural contraction (4), and mediates the vasodilator response to acetylcholine, the cholinergic neural transmitter (4), as well as the vasodilator responses to vasoactive humoral substances (4).
The central role of NF-κB in septic pathologies and the predominant influence of endothelium on vascular homeostasis suggest that activation of endothelial specific NF-κB signaling may play an essential role in the development of septic shock and septic vascular dysfunction. However, the causative role of endothelial intrinsic NF-κB in septic shock has not been studied. A number of studies have examined endothelial NF-κB activation and its role in endothelial inflammation (21–24). However, those studies were performed with cultured endothelial cells, and have not addressed the pathogenic role of endothelial NF-κB activation in septic shock and septic vascular dysfunction. Several previous studies, including our own, have shown that inhibition of NF-κB activation using chemical NF-κB inhibitors alleviates septic shock and septic vascular dysfunction (13–15). However, those inhibitors inhibit NF-κB activation in all cell types, and may have effects that are not related to NF-κB inhibition. The causal contribution of endothelial intrinsic NF-κB to the pathogenesis of septic shock and septic vascular dysfunction has never been examined, due to lack of an investigative tool.
Recently, we have created and characterized transgenic mice designated as EC-TG mice, in which a mutant I-κBα (I-κBαmt), a specific inhibitor of NF-κB, is expressed in endothelial cells under the control of the tetracycline gene regulatory system (17). The EC-TG mice display endothelial-restricted blockade of NF-κB pathway (17) and enable us to selectively inhibit endothelial NF-κB activation in vivo under physiological setting. A preliminary study using those mice showed that endothelial NF-κB blockade partially reversed endotoxemic hypotension (17). The current study extends our preliminary study by examining the effects of selective blockade of endothelial NF-κB pathway on the cascades of molecular events that lead to septic shock and septic vascular dysfunction in LPS and cecal ligation and puncture (CLP) models of sepsis. We demonstrated that blockade of endothelial specific NF-κB signaling is sufficient to abrogate the molecular cascades leading septic vascular dysfunction. Our data defines the mechanistic role of endothelial intrinsic NF-κB in the pathogenesis of septic shock and septic vascular dysfunction, and provides new insights into the molecular mechanisms of sepsis and septic shock.
The generation and characterization of the EC-TG mice that conditionally overexpress I-κBαmt selectively on endothelium have been previously described (17). Here, we utilized this mouse strain to define the causative contribution of endothelial intrinsic NF-κB to septic shock and septic vascular dysfunction. We studied 8 groups of mice (8–10 weeks, on FVB genetic background): transgene negative control or sham (WT-Con, WT-sham), transgene negative LPS or CLP (WT-LPS, WT-CLP), TG control or sham (TG-Con, TG-sham) and TG LPS or CLP (TG-LPS, TG-CLP). We also studied 4 groups of mice on B6129S genetic background (from Jackson Laboratory, stock numbers, WT mice, 101045, TNF-α knockout, 003008): WT-Con, WT-LPS, TNF-α knockout control (TNF-KO-Con) and TNF-KO-LPS. All animal experiments were approved by the institutional animal care and use committee and complied with NIH Guide.
Mice were anesthetized with tribromoethanol (300 mg/kg, i.p.), intubated and ventilated with a mouse ventilator as we have previously described (13). We chose to use tribromoethanol as anesthetics because it causes less cardiovascular depression (25). A micro-cannula was inserted into carotid artery for continuously monitoring systemic blood pressure. Mouse body temperature was kept constant with a servo controlled electronic blanket and intra-anal thermal probe. After a 30-minute equilibration period and measurement of basal blood pressure, mice were injected with saline or LPS (E. Coli 0111:B4, 2.5 mg/kg, i.p.). Systemic blood pressure was recorded for 4 hours, and mean arterial blood pressure (MBP) calculated. In a separate set of experiments, mice were injected with saline or LPS (10 mg/kg, i.p.). At 24 hours after saline or LPS injection, systemic blood pressure was recorded as described above.
For the CLP model, mice were anesthetized and cannulated at 18 hours after operation, and systemic blood pressure was recorded as described above.
Mice were anesthetized and cannulated at 5.5 hours after saline or LPS (10 mg/kg, i.p.) injection. Because basal blood pressure influences vascular reactivity, mice that had low initial MBP were resuscitated with 6% dextran in 7.5% NaCl during the equilibration period to ensure a comparable baseline MBP among all groups. Following the measurement of baseline MBP, dose-response relationship to α-adrenergic receptor agonist, norepinephrine (NE, 30, 100, and 300 ng/kg, i.v. bolus injection), to the endothelium-dependent vasodilator, acetylcholine (Ach, 60, 200 and 600 ng/kg, i.v. bolus injection), or to the endothelium-independent vasodilator, sodium nitroprusside (SNP, 60, 200 and 600 ng/kg, i.v. bolus injection) was recorded in three separate sets of experiments. The maximum increase, or decrease in MBP elicited by each dose of NE, or Ach or SNP was calculated and compared.
At 6 hours after saline or LPS (10 mg/kg, i.p.) injection, the mesenteric vascular bed was isolated, as previously described (26), and perfused with oxygenated physiological salt solution at a constant flow rate of 200 μl/min. Because perfusion flow rate is constant, changes in perfusion pressure represent changes in vascular resistance. Following a 40-min equilibration period, dose-response to NE (30, 100 and 300 ng) was recorded. To study vasodilator response, perfusion pressure was elevated by approximately 60 mmHg by perfusing the mesenteric vascular beds with 100 μM NE. After a sustained elevation in perfusion pressure has achieved, dose-response to Ach or SNP (1, 10 and 100 ng) was recorded in two separate sets of experiments. Ach or SNP was injected into the perfusion circuit immediately proximal to the mesenteric artery. The maximal increase or decrease in perfusion pressure caused by each dose of NE, or Ach or SNP was calculated and compared.
Mice in sham groups were subjected to sham, and in CLP groups subjected to CLP operation as we have previously described (17) using an 18-gauge needle. At 18 hours post-operation, mice were cannulated for systemic blood pressure measurement as described above.
Aortic levels of iNOS and eNOS proteins were determined by Western blot as we previously described (17) using antibodies against iNOS, eNOS and actin (all from Santa Cruz).
Plasma and aortic levels of nitrite/nitrate, the stable metabolic product of NO, were measured using nitrite/nitrate assay Kit (Cayman Chemical). Aortic level of TNF-α was determined using ELISA Kit (eBIOscience).
Cryosections (6 μm) were prepared from aorta of each group of mice at 6 hours after saline or LPS injection, fixed with paraformaldehyde, permeabilized, blocked with blocking solution, and incubated with rabbit anti-TNF-α antibody (Abcam Inc, MA) or rabbit anti-iNOS antibody (Santa Cruz) overnight at 4°C. Specific binding was detected with biotinylated secondary antibody-horseradish peroxidase complexes using VECTASTAIN® Elite ABC kits (Vector Laboratories). Antigen-antibody complexes were visualized using 3′, 3′-diamino benzidine (Vector Laboratories). Sections were counterstained with hematoxylin, mounted and viewed under light microscope.
Data were expressed as mean ± S.E.M, and analyzed using ANOVA or Kruskal-Wallis Rank test, followed by Holm-Sidak method or Dunnett’s test for post hoc analysis. The null hypothesis was rejected at 5% level.
To define the role of endothelial intrinsic NF-κB signaling in septic shock, WT-Con, TG-Con were injected with saline and WT-LPS and TG-LPS mice were injected with LPS. Mean systemic arterial blood pressure (MBP) was monitored for 4 hours. Baseline MBP was identical in the 4 groups of mice and decreased slightly over time, likely due to loss of blood or body fluids (Fig. 1A). At 3 and 4 hours post-LPS, WT-LPS mice showed a marked drop in MBP, which was significantly less in TG-LPS (Fig. 1A). The effect of endothelial NF-κB blockade on the development of septic shock was further examined at late time points in both LPS and CLP models of sepsis. Compared with control or sham group of mice, WT-LPS or WT-CLP mice showed a significant drop in MBP at 24 hours after LPS injection (Fig. 1B) or at 18 hours after CLP operation (Fig. 1C), which was significantly attenuated or prevented in TG-LPS or TG-CLP mice (Figs. 1B and 1C). These results unveil an important role for endothelial specific NF-κB signaling in the development of septic shock.
A major feature of septic vascular dysfunction is the repressed vasoconstrictor response to catecholamine. We have therefore examined whether endothelial selective NF-κB blockade alters the pressor response to NE in control and endotoxemic mice. Because basal vascular tone affects vascular reactivity, mice with low initial MBP were resuscitated to ensure comparable levels of MBP among all groups of mice before starting the NE trial. Baseline MBP was 94±1, 91±2, 93±1 and 93±1 mmHg for WT-Con, WT-LPS, TG-Con and TG-LPS group, respectively. As expected, NE caused a dose-dependent elevation in MBP. Compared with that in WT-Con and TG-Con mice, the NE-elicited elevation in MBP decreased significantly in WT-LPS at all 3 doses (Fig. 2A). In contrast, the NE-elicited elevation in MBP in TG-LPS mice was comparable to that in WT-Con and TG-Con mice, and was significantly higher than that in WT-LPS mice (Fig. 2A).
In vivo vasoreactivity is affected by systemic factors such as cardiac output and reflex. To avoid these effects, we further assessed the NE response in isolated perfused mesenteric vascular beds. Because the vascular bed was perfused at constant flow rate, changes in perfusion pressure represent alteration in vascular resistance. Mesenteric perfusion pressure was comparable among the 4 groups of mice at baseline, but was elevated by NE injection in a dose-dependent manner (Fig. 2B). Compared with WT-Con and TG-Con mice, the NE-elicited elevation in mesenteric perfusion pressure was greatly attenuated in WT-LPS mice (Fig. 2B), but was not affected in TG-LPS mice (Fig. 2B). These results indicate that blockade of endothelial intrinsic NF-κB abrogates LPS-induced repression of the vasoconstrictor response to NE, suggesting that activation of endothelial intrinsic NF-κB pathway plays an important role in the development of vascular hyporesponsiveness to NE in endotoxemic mice.
We next examined whether endothelial selective NF-κB blockade prevents the impairment of endothelium-dependent vasodilator response, another major feature of septic vascular dysfunction. Mice were injected with saline or LPS, cannulated and resuscitated as described above. Baseline MBP was comparable among the 4 groups of mice before initiation of Ach or SNP trial. Both Ach (endothelium-dependent vasodilator) and SNP (endothelium-independent vasodilator) caused dose-dependent drop n MBP (Figs 3A and 3B). Compared with WT-Con and TG-Con mice, WT-LPS mice displayed a significantly blunted vasodilator response to Ach at all 3 doses (Fig. 3A). In contrast, TG-LPS mice showed an Ach response that was identical to that of WT-Con and TG-Con mice, and was significantly bigger than that of WT-LP mice (Fig. 3A). The 4 groups of mice showed a similar vasodilator response to SNP (Fig. 3B).
Likewise, Ach and SNP caused dose-dependent drop in perfusion pressure in the isolated perfused mesenteric vascular bed (Figs 4A and 4B). The drops in perfusion pressure evoked by the 3 doses of Ach, but not by the 3 doses of SNP, were significantly less in WT-LPS mice as compared to that of WT-Con and TG-Con mice (Figs 4A and 4B). In TG-LPS mice, Ach or SNP caused a drop in mesenteric perfusion pressure that was similar to that in WT-Con and TG-Con mice, and the Ach-mediated response was significantly bigger than that of WT-LPS mice (Figs 4A and 4B). Overall, these results illustrate that selective blockade of endothelial NF-κB pathway prevents the LPS-induced impairment of endothelium-dependent vasodilator response to Ach, implying that activation of endothelial intrinsic NF-κB plays an important role in the impairment of endothelium-dependent vasodilator response during endotoxemia.
LPS causes systemic hypotension and vascular hyporeactivity by inducing iNOS expression, resulting in overproduction of NO, which causes hypotension and blunts vasoconstrictor response (5–7). To investigate the mechanism through which endothelial selective NF-κB blockade restores systemic MBP and vascular reactivity, we determined aortic iNOS protein expression in WT and TG mice. Fig. 5A was Western blot photographs showing that endothelial selective NF-κB blockade inhibited LPS-induced iNOS protein expression in aortae. The iNOS bands were quantified using densitometry and summarized in Fig. 5B. Aortic level of iNOS protein was negligible in WT-Con and TG-Con mice, increased markedly in WT-LPS mice, but reduced by approximately 73% in TG-LPS mice, as compared with that in WT-LPS mice (Fig. 5).
Endothelial selective NF-κB blockade also inhibited iNOS activity as indicated by the reduced plasma and aortic levels of nitrite/nitrate in TG-LPS mice (Figs 6A and B). Compared with WT-Con and TG-Con mice, WT-LPS mice showed an approximately 5-fold increase in plasma level and 6-fold increase in aortic level of nitrite/nitrate, which were reduced by approximately 64% and 52% in TG-LPS mice (Fig. 6). Thus, blockade of endothelial intrinsic NF-κB suppresses aortic iNOS expression and activity, suggesting that endothelial selective NF-κB blockade prevents systemic hypotension and vascular hyporeactivity by inhibiting iNOS expression in vascular tissues.
Ach causes vasodilatation by activating eNOS, resulting in the release of endothelium-derived NO (10, 11). We have therefore compared aortic levels of eNOS protein expression between WT-LPS and TG-LPS mice. Fig. 7A was Western blot photographs showing that endothelial selective NF-κB blockade abrogated LPS-induced eNOS protein down-regulation. Fig. 7B summarized the densitometry quantification of the eNOS bands. Consistent with impaired vasodilator response to Ach, WT-LPS mice showed a significantly reduced aortic expression of eNOS protein, as compared with WT-Con and TG-Con mice (Fig. 7). Aortic level of eNOS protein in TG-LPS mice was identical to that of WT-Con and TG-Con mice (Fig. 7). These results suggest that activation of endothelial specific NF-κB contributes to the LPS-induced down-regulation of eNOS expression, which accounts for the impairment of endothelium-dependent vasodilator response to Ach in endotoxemic mice.
To establish a link between activation of endothelial NF-κB signaling and eNOS down-regulation, we examined the effect of endothelial NF-κB blockade on aortic TNF-α expression, and determined the role of TNF-α in LPS-induced eNOS down-regulation in our mouse model. Compared with WT-Con and TG-Con mice, WT-LPS mice displayed a 10-fold increase in aortic level of TNF-α protein, which was reduced by approximately 51% in TG-LPS mice (Fig. 8). To define the role of TNF-α in LPS-induced eNOS down-regulation, we compared aortic levels of eNOS protein expression between TNF-α-KO mice and their genetic background matched WT mice at 6 hours after LPS injection. Compared with that in WT-Con and TNF-α-KO-Con mice, aortic eNOS expression was significantly down-regulated in WT-LPS mice, which was prevented in TNF-α-KO-LPS mice (Fig. 9). This result indicates that TNF-α plays an obligatory role in LPS-induced eNOS down-regulation. Collectively, these results suggest that activation of endothelial NF-κB signaling leads to eNOS down-regulation via TNF-α-dependent mechanisms.
We next performed immunohistochemical staining of aortic sections from WT and TG mice to identify the cells whose levels of iNOS and TNF-α expression was inhibited by endothelial selective NF-κB blockade. TNF-α- and iNOS-positive cells were not detectable in aortic sections from WT-Con and TG-Con mice (Figs. 10A, B, E. and F), markedly increased in aortic sections from WT-LPS mice (Figs. 10C and G) and significantly reduced in aortic sections from TG-LPS mice (Figs. 10D and H). Strong TNF-α and iNOS staining was localized to endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) in aorta of WT-LPS mice (Figs. 10C and G). In TG-LPS mice, TNF-α and iNOS-positive ECs were barely detected, and TNF-α and iNOS-positive VSMCs were significantly reduced (Figs. 10D and H). This result illustrates that endothelial selective NF-κB blockade diminishes EC TNF-α and iNOS expression, and also inhibits VSMC TNF-α and iNOS expression.
The major finding of this study is that endothelial-specific NF-κB signaling plays a pivotal role in the pathogenesis of septic shock and septic vascular dysfunction. Challenge of WT mice with LPS resulted in a marked drop in systemic MBP at both early and late time points, a significantly blunted vasoconstrictor response to NE and an impaired endothelium-dependent vasodilator response to Ach. WT mice underwent CLP also showed a significant systemic hypotension. Consistent with the changes in physiological parameters, WT-LPS mice showed a markedly increased aortic iNOS protein expression, elevated plasma and aortic levels of nitrite/nitrate, the stable end-products of NO, increased aortic TNF-α protein expression and decreased aortic eNOS protein expression. In sharp contrast to WT-LPS mice, TG-LPS mice with endothelial selective NF-κB blockade displayed no significant hypotension, a normal vasoconstrictor response to NE, no impairment in the endothelium-dependent vasodilator response to Ach, reduced aortic iNOS expression, decreased plasma and aortic levels of nitrite/nitrate, reduced aortic TNF-α expression and increased aortic eNOS expression. TG-CLP mice were also prevented from systemic hypotension. Taken together with our previous demonstration that the EC-TG mice express the NF-κB inhibitor, I-κBαmt, only on endothelium and display endothelial selective blockade of NF-κB activation (17), these results illustrate that blockade of endothelial intrinsic NF-κB pathway mitigates the cascade of molecular events that lead to septic shock and septic vascular dysfunction, implying that endothelial specific NF-κB signaling plays a pivotal role in the development of septic shock and septic vascular dysfunction.
Several groups, including our own, have shown that inhibition of NF-κB activation reduces systemic hypotension and restores or partially restores vascular reactivity in mice or rats subjected to endotoxemia or multimicrobial sepsis (13–15). However, those studies used non-specific chemical NF-κB inhibitors, which may have non-specific effects. More importantly, those studies inhibited NF-κB in all cell types, and did not address the function of cell specific NF-κB in the pathological processes. This study is the first to define the specific contribution of endothelial intrinsic NF-κB signaling to septic shock and septic vascular dysfunction, and thus provides novel insights into the molecular mechanisms of septic shock and septic vascular dysfunction.
It is well documented that LPS or sepsis causes systemic hypotension and vascular hyporesponsiveness by activating NF-κB-mediated iNOS expression (2, 3), leading to an excessive production of NO. The marked inhibition of LPS-induced iNOS expression and activity by selective blockade of endothelial NF-κB pathway with a concomitant abrogation of LPS-induced hypotension and vascular hyporeactivity supports the notion that endothelial NF-κB blockade alleviates septic hypotension and septic vascular dysfunction by inhibiting NF-κB-mediated iNOS expression.
We showed that LPS impaired endothelium-dependent vasodilator response in WT-LPS, but not in TG-LPS mice. The preservation of endothelium-dependent vasodilator response in TG-LPS mice could be explained by the prevention of TNF-α mediated eNOS down-regulation. First, TNF-α is a NF-κB-regulated gene product (2, 3), and blockade of endothelial NF-κB significantly inhibited LPS-induced TNF-α expression in TG-LPS mice. Secondly, TNF-α is known to down-regulate eNOS expression (8). We demonstrated in current study that TNF-α mediates LPS-induced eNOS down-regulation. Thirdly, it is well documented that Ach elicits vasodilatation by stimulating eNOS-mediated endothelial NO release (4, 10, 11). Fourthly, we demonstrated here that blockade of endothelial NF-κB concomitantly inhibited the LPS-induced TNF-α up-regulation and eNOS down-regulation in vascular tissue, and restored endothelium-dependent vasodilatation. Inhibition of endothelial NF-κB activation suppresses local inflammation within endothelial cells, which could reduce the production of reactive oxygen species and increase NO bioavailability (27). This may also contribute to the preserved endothelium-dependent vasodilator response to Ach in TG-LPS mice.
Physiologically, vascular tone is mainly influenced by the physical and biochemical properties of vascular smooth muscle cells. NF-κB pathway in smooth muscle cells is not inhibited in our EC-TG transgenic mice (17). The effectiveness of endothelial-restricted NF-κB inhibition in correcting septic vascular dysfunction and in inhibiting aortic iNOS expression is somewhat unexpected. Endothelial-selective inhibition of NF-κB activation restored systemic MBP to 80% of control level, completely abrogated LPS-induced repression of vasoconstrictor responses to NE, and inhibited LPS-induced aortic iNOS protein expression by approximately 73%. These results highlight the importance of endothelium and endothelial specific NF-κB signaling in septic shock and septic vascular dysfunction. These results suggest that cross talk between ECs and VSMCs may be an important mechanism regulating inflammatory response within vascular wall during sepsis. In support of this speculation, we demonstrated here that selective blockade of endothelial NF-κB pathway not only diminished LPS-induced TNF-α and iNOS expression in ECs, but also significantly inhibited TNF-α and iNOS expression in VSMCs. Further studies to elucidate the mechanisms underlying the paracrine interactions between ECs and VSMCs will help to better understand the molecular mechanisms of vascular wall inflammation during sepsis and other pathological conditions.
Our current results are in good agreement with our previous studies demonstrating that endothelial selective NF-κB blockade ameliorated multiple organ injury (MOI) and improved survival in septic mice (17). Our results are also consistent with two recent reports showing that endothelial-specific NF-κB suppression attenuated hypertension-induced renal damage (28) and high fat diet-induced atherosclerosis (29). It is reported that blockade of endothelial NF-κB pathway enhanced LPS-induced endothelial permeability (30). This result does not necessarily contradict our findings, because different models (conventional versus conditional transgenic mice) were used in the two studies. The two mouse models have different basal endothelial barrier integrity and, therefore, display different response to LPS in term of alteration in endothelial permeability (17, 30). Nevertheless, both studies illustrate a critical role of endothelial NF-κB in controlling endothelial barrier integrity and function.
Our findings have therapeutic implications. The central roles of NF-κB in systemic inflammation and in septic pathology indicate that NF-κB is an ideal target for therapeutic intervention (2, 12–16). However, NF-κB inhibition impairs host defense mechanism and causes immune suppression (31, 32). Consequently, the beneficial anti-inflammatory effect can be offset by the detrimental pro-infectious effect of NF-κB inhibition, leading to unaltered or even worse outcome. To overcome this problem, we need to develop novel approaches that can selectively inhibit NF-κB-mediated inflammatory and injurious responses (detrimental) without significantly interfering with the NF-κB-mediated immune and host defense responses (beneficial). Our current and previous demonstration that selective blockade of NF-κB-driven inflammatory response within endothelium ameliorated septic MOI (17), abrogated septic vascular dysfunction and improved survival, but had no effect on bacterial clearance capacity (17) provides experimental basis for endothelial selective NF-κB inhibition as an innovative strategy to develop sepsis therapies.
We would like to thank Dr. J. W. Pollard and staff at AECOM Transgenic and Gene Targeting Facility for help in generating transgenic mice.
This work was supported by NIH grant GM063907 and the Faculty Award Program of the Feinstein Institute for Medical Research.
The authors have no financial conflict of interest.