MECHANISTIC ROLE OF THE CALCIUM-DEPENDENT PROTEASE CALPAIN IN THE ENDOTHELIAL DYSFUNCTION INDUCED BY MYELOPEROXIDASE

Abstract

Myeloperoxidase (MPO) is a peroxidase enzyme secreted by activated leukocytes that plays a pathogenic role in cardiovascular disease, mainly by initiating endothelial dysfunction. The molecular mechanisms of the endothelial damaging action of MPO remain though largely elusive. Calpain is a calcium-dependent protease expressed in the vascular wall. Activation of calpains has been implicated in inflammatory disorders of the vasculature. Using endothelial cells and genetically modified mice, this study identifies the μ-calpain isoform as novel downstream signaling target of MPO in endothelial dysfunction. Mouse lung microvascular endothelial cells were stimulated with 10 nmol/L MPO for 180 min. MPO denitrosylated μ-calpain C-terminus domain, and time-dependently activated μ-calpain, but not the m-calpain isoform. MPO also reduced Thr¹⁷² AMPK and Ser¹¹⁷⁷ eNOS phosphorylation via upregulation of PP2A expression. At the functional level, MPO increased endothelial VCAM-1 abundance and the adhesion of leukocytes to the mouse aorta. In MPO treated endothelial cells, pharmacological inhibition of calpain activity attenuated expression of VCAM-1 and PP2A, and restored Thr¹⁷²AMPK and Ser¹¹⁷⁷eNOS phosphorylation. Compared to wild-type mice, μ-calpain deficient mice experienced reduced leukocyte adhesion to the aortic endothelium in response to MPO. Our data first establish a role for calpain in the endothelial dysfunction and vascular inflammation of MPO. The MPO/calpain/PP2A signaling pathway may provide novel pharmacological targets for the treatment of inflammatory vascular disorders.

Summary

Our study identifies endothelial calpain as a new molecular target of myeloperoxidase, thus helping explain its detrimental cardiovascular actions. We also first report that increased calpain activity downregulates important endothelial cell functions thus causing adhesion of circulating white blood cells to the vascular wall. Our study might provide new molecular targets in the treatment of the many acute and chronic vascular disorders associated with inflammation.

Introduction.

Preclinical studies indicate that circulating cells of the myeloid compartment, namely neutrophils and monocytes, play an important role in the initiation and progression of vascular disease¹. In humans, activation of neutrophils and monocytes are highly associated with cardiovascular events^{2, 3}. Accordingly, epidemiological studies have associated leukocyte with cardiovascular disease⁴, which underlines their role in low-grade inflammatory disorders of the vasculature.

Myeloperoxidase (MPO) is a major neutrophil effector protein, accounting for approximatively 5% of the cell’s dry mass, and it is also present in monocytes, where it comprises one-third of the MPO content found in neutrophils. MPO and its byproducts have been shown to contribute to endothelial dysfunction⁵, hypertension⁶, and rupture of atherosclerotic plaques⁷. Plasma and serum levels of MPO independently predict endothelial dysfunction of large arteries in cardiovascular patients^{5, 8}, and individuals who have higher MPO levels are 15- to 20-fold more likely to demonstrate abnormal coronary angiograms⁹. Conversely, a promoter polymorphism associated with a twofold reduction in MPO expression protects from vascular disease and cardiac death¹⁰. Surprisingly, very little information is available in the scientific literature to explain the molecular mechanism(s) through which MPO impairs endothelial function, which limits therapeutic intervention in humans.

Calpain is a Ca²⁺ dependent, cysteine protease¹¹ whose activity is regulated, among other factors, by its endogenous inhibitor, calpastatin¹². Two calpain isoforms, μ- and m- calpain, are expressed in endothelial cells¹³, where they have been shown to affect nitric oxide generation¹⁴ and endothelial adhesiveness to leukocytes¹⁵. Moreover, inhibition of calpain attenuates endothelial dysfunction in acute¹⁶ and chronic¹⁷ inflammatory states of the cardiovascular system. We hypothesized a role for calpain in the endothelial dysfunction induced by MPO. Using pharmacological tools and genetically modified mice, we investigated the mechanism(s) through which MPO activates endothelial calpain. We also studied the molecular targets and functional consequences of MPO/calpain signaling in the mouse aortic endothelium.

Material and Methods

The data, analytical methods, and study materials that support the findings of this study are available from the corresponding author upon reasonable request Detailed Methods are available in the online-only Data Supplement. This study was performed in accordance with the NIH and Temple University IACUC guidelines for the use of experimental animals. Eight to twelve-week-old wild-type C57BL/6J mice (Stock #002682, Jackson Laboratory, Ann Arbor MI), and mice deficient in μ-calpain (Capn1^−/−; stock number EM: 02362; European Mouse Mutant Archive (http://www.emmanet.org/strains.php) were used. All mice were male with an average body weight of 24 grams. Mice were used to study the effect of MPO/calpain signaling on leukocyte-endothelium interaction in the aorta, according to a previously published method¹⁸, further described in the online-only Data Supplement. Systemic activation of circulating leukocytes occurs in the microcirculation where, following interaction with endothelial cells, leukocyte become primed for degranulation¹⁹. Accordingly, we used mouse lung microvascular endothelial cells (MMVEC).

Statistical Analyses.

Results are expressed as mean±SEM. Two-way ANOVA (with Bonferroni’s correction for comparison of multiple means) was used for comparisons of calpain time course activity in cells exposed to MPO (Figure 1, upper panel). One-way ANOVA (with Bonferroni’s correction for comparison of multiple means) was used for comparisons of leukocyte adhesion assays and protein expression and modification studies. A Students Paired t test was used to compare the two experimental groups in Figure 3. P values < 0.05 were considered significant. All statistical evaluations were performed using Prism version 7.0d for Apple, GraphPad Software, La Jolla California USA, www.graphpad.com”.

Figure 1. MPO activates μ-calpain in endothelial cells.

Upper panel: Serum starved MMVEC were incubated with 10 nmol/L MPO and calpain activity was measured at 60, 120, 180, and 240 min using the calpain specific fluorogenic substrate Succ-LLVY-AMC. Lower panels: MMVEC were then incubated with either vehicle (8 μL PBS) or 10 nmol/L MPO for 180 min with or without ZLLal (100 μmol/L - 30 min pretreatment). The activity of the μ-calpain isoform was assessed by immunoblot analysis using a primary antibody that recognizes cleavage of the N-terminus domain of μ-calpain large subunit. Loss of N-terminus domain indicates calpain activation. An antibody against μ-calpain large subunit C-terminus domain was used to quantify total μ-calpain expression, β-Actin detection was used as a loading control. Representative immunoblot demonstrate cleavage of MMVEC μ-calpain N-terminus domain following incubation with 10 nmol/L MPO for 180 min. Bar graph summarizes densitometry quantification of μ-calpain N-terminus domain. Total μ-calpain expression level was not changed by MPO, as demonstrated C-terminus domain immunoblot. Data are mean±SEM of 4 (upper graph) and 6 to 8 (lower graph) independent experiments.

Figure 3. MPO induces denitrosylation of μ-calpain.

Serum starved MMVEC were incubated with vehicle (8 μL PBS) or 10 nmol/L MPO for 180 min. Nitrosylation levels of μ-calpain in MMVEC was assessed by biotin switch assay. μ-Calpain was detected in the streptavidin-purified mixture using standard immunoblot techniques. Data are mean±SEM of 5 independent experiments.

Results

MPO Activates the Endothelial μ-Calpain Isoform.

Data shown in Figure 1 (upper panel) demonstrate that exposure of MMVEC to 10 nmol/L MPO causes a time-dependent increase in calpain activity, with near maximum activity reached after 180 min. The MPO concentration used in our study was based on the results of dose-response curves (supplemental Figure S2), and it is in the range of MPO plasma levels found in humans with coronary artery disease²⁰. To identify the calpain isoforms activated by MPO, we then measured expression levels of the N-terminus domain of the 80 kDa subunit of both m- and μ-calpain. Upon activation, calpains undergo autoproteolysis with removal of the 9-15 amino acids of the N-terminus domain. Cleavage of the N-terminus domain was the detected by immunoblot analyses as loss of N-terminus domain recognition (see also method section). MMVEC exposed to MPO experience cleavage of the N-terminus domain of μ-calpain (Figure 1, lower panel and supplemental figure S1A), but not that of m-calpain (supplemental Figure S3). To rule out nonspecific degradation of μ-calpain by MPO, we quantified total μ-calpain expression using a primary antibody against domain IV of the large subunit, which recognizes both unautolyzed and autolyzed μ-calpain. As shown in Figure 1 (lower panel) MPO did not cause significant changes in μ-calpain expression level. Similarly, μ-calpain mRNA levels were not significantly changed by MPO (supplemental Figure S4). In parallel experiments, the activating effect of MPO on μ-calpain was blocked by pretreatment of MMVEC with 100 μmol/L ZLLal, a selective calpain inhibitor¹⁷. Data shown in Figure 1, lower panel, demonstrate that ZLLal effectively prevented calpain activation in response to MPO. Taken together, these data demonstrate that MPO activates μ-calpain in endothelial cells, and that this process is amenable to pharmacological inhibition.

The Mechanism(s) Through Which MPO Activates μ-Calpain.

To investigate the mechanism(s) responsible for μ-calpain activation, we first measured the effect of MPO on calpastatin, which is the endogenous inhibitor of calpain¹¹. Figure 2 and supplemental Figure S1B show reduced calpastatin abundance in MMVEC exposed to 10 nmol/L MPO. At first, this result suggested that degradation of calpastatin by MPO could be the mechanism responsible for activation of μ-calpain. Data in the literature though have reported loss of calpastatin in the presence of active calpains, due to the fact that calpastatin itself is a calpain substrate¹¹. Accordingly, we further studied whether the loss of calpastatin in MPO treated MMVEC was secondary to calpain activation. Indeed, MPO failed to reduce calpastatin levels in MMVEC pretreated with the calpain inhibitor ZLLal (Figures 2 and S1B), thus confirming that, under our experimental conditions, loss of calpastatin is secondary to, and due to μ-calpain activation. Consistent with these results calpastatin mRNA expression levels were also not significantly changed (supplemental Figure S4). These data indicate that MPO alters the calpain/calpastatin balance in endothelial cells, by causing sustained activation of μ-calpain with subsequent degradation of calpastatin.

Figure 2. MPO causes degradation of calpastatin via calpain activation.

Serum starved MMVEC were incubated with vehicle (8 μl PBS) or 10 nmol/L MPO for 180 min with or without ZLLal (100 μmol/L, 30 min) pretreatment. The expression of the endogenous calpain inhibitor calpastatin was measured by immunoblot analysis and quantified by densitometry. Beta actin detection was used as a loading control. Data are mean±SEM of 6 to 7 independent experiments.

We then tested whether MPO activates calpain via a nitrosylation mediated mechanism. Studies have shown that nitric oxide inactivates calpains by nitrosylation²¹, and that denitrosylation increases calpain activity²². Therefore, we measured μ-calpain nitrosylation levels in MMVEC exposed to MPO using a previously described biotin switch assay²³. As shown in Figure 3 MPO reduces μ-calpain nitrosylation levels in MMVEC. These data suggested that perturbation in the eNOS/NO biosynthetic machinery is a probable mechanism through which MPO activates the endothelial calpain system.

μ-Calpain plays a Role in the Mechanism through which MPO Decreases AMPK/eNOS Phosphorylation.

Phosphorylation of eNOS at Ser¹¹⁷⁷ is required for maintaining physiological levels of nitric oxide in the vascular endothelium²⁴ Thus, we tested the hypothesis that MPO impairs Ser¹¹⁷⁷ eNOS phosphorylation thereby sustaining calpain denitrosylation. Data shown in the upper panel of Figure 4, demonstrate that MPO significantly decreases Ser¹¹⁷⁷ eNOS phosphorylation in MMVEC in the absence of significant changes in total eNOS expression. Studies have demonstrated that in endothelial cells the energy sensing kinase AMPK activates eNOS through Ser¹¹⁷⁷ phosphorylation²⁵. Interestingly, studies have shown that calpain can affect AMPK expression and phosphorylation of its Thr¹⁷² activation site²⁶. Data shown in Figure 4, lower panel, demonstrate a significant decrease in AMPK Thr¹⁷² phosphorylation whereas total AMPK expression remained unchanged in MPO exposed MMVEC. Treatment of MMVEC with the calpain inhibitor ZLLal restored AMPK and eNOS phosphorylation in the presence of MPO (Figure 4, and supplemental Figures S1C and S1D), which suggests the existence of a feed-forward control mechanism through which in response to MPO active calpains maintain a denitrosylated status by sustaining downregulation of AMPK/eNOS signaling. Akt also activates eNOS via Ser¹¹⁷⁷ phosphorylation²⁴. Therefore, we also measured Akt activity with Ser⁴⁷³Akt phosphorylation in response to MPO, which was found to be reduced by MPO in a non calpain-dependent manner (supplemental Figures S5 and S1K).

Figure 4. MPO/calpain signaling downregulates eNOS/AMPK phosphorylation.

Serum starved MMVEC were incubated with vehicle (8 μL PBS) or 10 nmol/L MPO for 180 min with or without ZLLal (100 μmol/L, 30 min) pretreatment. Phosphorylation levels of Ser¹¹⁷⁷ eNOS and Thr¹⁷² AMPK, in MMVEC were assessed by immunoblot analysis. Data are mean±SEM of 6 to 8 independent experiments.

The Mechanism through which MPO/Calpain Signaling Affects Thr¹⁷² AMPK Phosphorylation.

First, we studied the effect of MPO on the liver kinase B1 (LKB1), which is the main kinase to phosphorylate AMPK at Thr^{172 27} MPO did not significantly changed LKB1 expression levels in MMVEC under our experimental conditions (Figures 5, upper panel and S1E). Second, we investigated the role of the protein phosphatase 2A (PP2A), a signaling partner of active calpains²⁸ that has also been shown to inhibit AMPK activity via Thr¹⁷² dephosphorylation²⁹. Interestingly MPO increased the expression level of the catalytic subunit of PP2A in MMVEC (Figures 5, middle panel, and S1F). In agreement with the data on AMPK/eNOS phosphorylation summarized in Figure 4, the effect of MPO on PP2A were abolished by treatment of MMVEC with ZLLal (Figures 5, middle panel, and S1F). To obtained mechanistic evidence that the effect of MPO/calpain signaling on AMPK phosphorylation is indeed PP2A dependent, we pretreated MMVEC with the PP2A inhibitor okadaic acid. Results shown in the lower panel of Figure 5 and in Figure S1G demonstrate that direct inhibition of PP2A preserves phospho-AMPK levels in MMVEC in the presence of MPO²⁹. These data uncover PP2A as an important downstream mediator of endothelial dysfunction in response to MPO/calpain signaling.

Figure 5. Role of LKB1 and PP2A in the effect of MPO/calpain signaling on AMPK phosphorylation.

Serum starved MMVEC were incubated with vehicle (8 μL PBS) or 10 nmol/L MPO for 30-180 min (LKB1 detection). Serum starved MMVEC were incubated with vehicle (X microL PBS) or 10 nmol/L MPO for 180 min (PP2A and pAMPK detection). with or without ZLLal (100 μmol/L, 30 min) pretreatment. Expression levels of LKB1 and PP2A in MMVEC were assessed by immunoblot analysis. Data are mean±SEM of 3 or 4 independent experiments for upper or lower panel, respectively.

Mechanistic Role of Calpain in the MPO-induced Increased Endothelial Adhesiveness to Leukocytes.

To investigate the impact on MPO/calpain signaling on endothelial function we measured the expression level of the endothelial cell adhesion molecule VCAM-1, an adhesion molecule upregulated by dysfunctional endothelia with impaired eNOS function³⁰. VCAM-1 expression levels are elevated in MMVEC cells treated with MPO (Figures 6, upper panel and S1H). Consistent with the biochemical data reported above, VCAM-1 protein levels were attenuated by treatment with ZLLal.

Figure 6. MPO increases endothelial adhesiveness to leukocytes.

Upper panels: Serum starved MMVEC were incubated with vehicle (8 μM PBS) or 10 nmol/L MPO for 180 min with or without ZLLal (100 μmol/L, 30 min) pretreatment Expression levels of VCAM-1 in MMVEC were assessed by immunoblot analysis. Lower panel: Circulating leukocytes and thoracic aortas were isolated from C57BLK and μ-calpain^−/− donor mice. Leukocytes were fluorescently labeled as reported in the method section. Isolated 2 mm length aortic segments were first exposed to 10 nmol/L MPO for 180 min. Aortic segments where then washed and co-incubated endothelial surface up with leukocytes for an additional 60 min. In parallel experiments, inhibition of calpain activity by ZLLal also prevented adhesion of leukocytes to the wild-type aorta exposed to MPO. Data are mean±SEM of 6 aortic segments from 3 mice per group. A total of 54 aortic segments were studied.

Endothelia expressing VCAM-1 becomes adhesive to circulating leukocytes ³¹. Accordingly, we next studied the effect of MPO/calpain signaling on the adhesion of leukocytes to the aortic endothelium. For these studies aortas and leukocytes were isolated from male wild-type/control C57BL/6J and μ-calpain deficient mice (Capn1^−/−). Lack of calpain expression was confirmed by western blot analyses (supplemental Figure S6). No significant differences in body weight, systemic blood pressure (supplemental Table S1), and total expression levels of eNOS and AMPK (Figure S6), were detected between wild-type mice and Capn1^−/− mice.

Data in Figure 6 show that in wild-type mice MPO increases the adhesion of leukocytes to the aortic endothelium. Compared to control aortas, MPO-stimulated aortas showed a significantly high number of adhered leukocytes, which was prevented by treatment of aortas with ZLLal (Figure 6, lower graph). Additional mechanistic studies with Capn1^−/− mice were undertaken to dissect the individual contribution of endothelial-expressed and/or leukocyte-expressed μ-calpain. As shown in Figure 6, lower panel, MPO fails to increase the adhesion of leukocytes isolated from wild-type mice to the aortic endothelium of μ-calpain deficient mice (Figure 6, lower graph). In contrast, increased leukocyte adhesion by MPO was observed in wild-type mouse aortas incubated with Capn1^−/− mouse leukocytes. Thus, endothelial expressed μ-calpain is a requirement for MPO to increases leukocyte-endothelium interactions. These data agree with data on VCAM-1 expression and they demonstrate that in the setting of increased MPO signaling, the vascular endothelium develops a calpain-dependent, pro-adhesive phenotype to leukocytes.

Discussion

The current study is, to our knowledge, the first to uncover the role of the cytosolic protease μ-calpain in the endothelial dysfunction associated with elevated levels of myeloperoxidase. We show that a concentration of MPO in the range of those found in the plasma of humans with cardiovascular disease activates the μ-calpain isoform in endothelial cells, which induces an adhesive phenotype in the mouse aorta. Noteworthy, we also show that activation of μ-calpain by MPO is amenable to pharmacological inhibition, which makes the endothelial calpain system an attractive, novel therapeutic target in cardiovascular disease.

Clinical studies have found a direct correlation between serum MPO levels and endothelial dysfunction; a correlation found to be stronger than that of C-reactive protein (CRP)⁵. Since MPO reacts with nitric oxide enhancing its catabolism⁸, current views consider direct interaction of MPO with nitric oxide as the primary mechanism underlying MPO-dependent alteration in endothelial function. This theory is based on seminal findings in animal models of sepsis demonstrating that MPO released by leukocytes localizes in and around endothelial cells of the aorta, where it drives catalytic consumption of nitric oxide³². This working model, though, does not address more recent studies demonstrating a direct inhibitory action of MPO and its derived metabolites on eNOS activity^{33, 34}. Our study uncovers a novel endothelial signaling mechanism, involving the calpain system, through which MPO decreases eNOS Ser¹¹⁷⁷ phosphorylation, thus causing endothelial dysfunction beyond catalytic consumption of nitric oxide (Figure S7 of online supplement). Thus, it is possible that following initial degradation of nitric oxide, more stable signaling alterations of eNOS take place in vascular endothelia exposed to MPO. The activation of calpain seen in our study could then help explain recent data reporting decreased eNOS function following exposure of endothelial cells to MPO byproducts³³. Our results also provide a molecular explanation to the evidence of increased nitric oxide bioavailability following calpain inhibition in cultured endothelial cells³⁵, and in animal models of vascular inflammation³⁶.

Several kinases regulate eNOS by phosphorylating its activation and/or inhibition sites. Noteworthy, phosphorylation at Ser¹¹⁷⁷, which is catalyzed by a number of distinct kinases including AMPK²⁵ and AKT³⁷ confers the most important positive modulation of eNOS activity, thereby increases NO production³⁸. In our study, MPO impaired the phosphorylation of both kinases, thus suggesting that binding of MPO to the vascular endothelium affects the activity of multiple kinases. Unique though to AMPK signaling, we found evidence that inhibition of calpain could restore both AMPK and eNOS Ser¹¹⁷⁷ phosphorylation in the face of MPO. We have also identified PP2A induction as a new molecular mechanism through which the MPO/calpain pathway downregulates AMPK. This result is consistent with evidence that AMPK alone can phosphorylate and activate eNOS at Ser^{1177 25}, and it also agrees with the prevailing theory that Ser¹¹⁷⁷ eNOS phosphorylation by diverse kinases is stimuli dependent and therefore regulated through multi-site phosphorylation in different pathologies³⁹. Of note, AMPK is an energy sensor/metabolic switch, because it phosphorylates and hence regulates the activity of enzymes such as acetyl-CoA carboxylase (ACC) and HMG-CoA reductase^{40, 41}. Interestingly, MPO has been recently shown to cause endothelial dysfunction in metabolic disorders such as hyperglycemia, obesity, and preeclampsia^42-45. Thus, it is possible that modulation of the calpain system may, at least in part, restore AMPK/eNOS signaling in the endothelial dysfunction of dysmetabolic states.

An unexpected, innovative result of our work is found in the mechanism through which MPO activates μ-calpain in the vascular endothelium. Our data demonstrate that reduced calpain nitrosylation occurs in endothelial cells exposed to MPO, which is in agreement with a recent study reporting a role for nitrosylation in the regulation of μ-calpain activity²². Others have also reported inhibition of calpain activity by nitric oxide^{46, 47}. Thus, a feedback regulation is likely to exists between the eNOS/NO biosynthetic machinery and the calpain system in the inflamed vascular endothelium. In the setting of inflammation, large amount of NO can be produced also by the inducible nitric oxide synthase isoform (iNOS), and data in the literature demonstrate that MPO can increase the catalytic activity of iNOS⁴⁸ in vitro. In contrast, others have demonstrated that, in the in vivo setting of inflammation, MPO downregulates iNOS expression⁴⁹. Induction of iNOS, however, requires a longer period of time then the 3 hour protocol adopted in this study⁵⁰, and therefore was not likely to play a significant role in this study.

Curiously, both calpain and eNOS require elevation in cytosolic calcium levels for activation, which in theory should consistently hinder physiological eNOS function since active calpain can downregulates eNOS⁵¹. This apparent paradox is explained, at least in part, by three lines of evidence. First, in response to physiologic agonist-stimulated calcium signaling binding of eNOS to HPS90 renders eNOS resistant to calpain digestion⁵². However, this physiological protective mechanism is likely to be impaired by MPO due to MPO ability to cause Ca²⁺ overload in endothelial cells⁵³, a phenomenon known to cause abnormal activation of calpain⁵⁴. Second, as demonstrated in this study MPO further downregulates eNOS function by reducing eNOS Ser¹¹⁷⁷ phosphorylation. Loss of Ser¹¹⁷⁷ phosphorylation reduces eNOS sensitivity to calcium⁵⁵, thus favoring the action of calpain. Third, our laboratory and others have demonstrated that sustained calpain activation disrupts HSP90/eNOS association ^56,57, which further reduces eNOS activity ⁵⁸.

In addition to its vasodilatory properties, basal levels of endothelial nitric oxide maintain an anti-adhesive phenotype in the vascular endothelium, thus preventing abnormal infiltration of leukocytes in the vascular wall^{30, 59}. Relevant here, endothelial nitric oxide has been shown to prevent upregulation of VCAM-1 in response to inflammatory stimuli³⁰. We found that MPO increases expression levels of VCAM-1 in microvascular endothelial cells, in addition to increasing adhesion of leukocytes to the mouse aorta via a calpain-dependent mechanism. In agreement with our results, others have demonstrated an association between MPO and increased leukocyte adhesion to the vascular endothelium⁶⁰, including the endothelium of the microcirculation⁶¹. Data also demonstrate that μ-calpain increases expression of cell adhesion molecules in endothelial cells⁶². Conversely, inhibiting calpain reduces the adverse outcomes of ischemia reperfusion injury by reducing ICAM-1 expression and preventing neutrophil infiltration⁶³. In previous studies we have also reported that inhibition of calpain activity prevents adhesion of leukocytes in the hyperglycemic microcirculation of diabetic rats¹⁷. Thus, inhibition of calpain attenuates adhesion of leukocytes to the vascular endothelium of both large conduit vessels and microcirculation which is in agreement with our in vitro and in vivo findings, respectively.

Interaction of circulating leukocytes with the vascular endothelium is governed by adhesion molecules expressed on the cell surface of both cell types. μ-Calpain is also constitutively expressed both in circulating leukocytes⁶⁴ and endothelial cells¹³. Thus, we asked whether leukocyte expressed μ-calpain played a role in these studies. We found evidence that aortas of mice deficient in μ-calpain are protected against leukocyte adhesion induced by MPO, which indicates an obligatory role for endothelial expressed calpains in the initiation of vascular inflammation in response to MPO. This conclusion is supported by data from our laboratory and others demonstrating that leukocyte-endothelium interactions can be largely abrogated by direct blockade of adhesion molecules expressed on the vascular endothelium only^{65, 66}. Nonetheless, it should be noted that endothelial expressed calpains have been shown to influence the adhesive and migratory behavior of circulating leukocytes⁶⁷. Conversely adhesion of leukocytes initiates calcium signaling events that lead to activation of the endothelial calpain system¹⁵. Obviously, further studies are needed to fully dissect how different inflammatory stimuli and/or duration of inflammation affect the calpain system of leukocytes and endothelial cells.

Perspectives

Preclinical and clinical research has indisputably confirmed the role of MPO in cardiovascular disease. The endothelium and eNOS are now well-established cellular and molecular targets of the cardiovascular damaging actions of MPO. Thus, MPO is an attractive target for therapeutic intervention in cardiovascular disease. Several selective MPO inhibitors have been developed in recent years. The necessity of preserving MPO immunodefensive functions, though, poses a major challenge to inhibition of MPO for therapeutic treatment. Perhaps, the uncovering of MPO downstream inflammatory pathways in relevant cells of the cardiovascular system will provide more selective pharmacological targets to hinder pathological activation of MPO, while preserving its beneficial host defense function. This study first identifies in endothelial-expressed calpain a new molecular target of MPO in the vascular wall thus providing new therapeutic targets. We also first report that increased calpain activity downregulate AMPK/eNOS signaling in endothelial cells via activation of the PP2A phosphatase, which questions the current working model that direct catalytic consumption or nitric oxide is the main mechanism through which MPO causes endothelial dysfunction. Furthermore, the implication of the energy sensing kinase, AMPK, in the endothelial dysfunction of MPO rises new intriguing questions on the role that MPO plays in the cardiovascular disease associated with the metabolic syndrome.

Novelty and Significance.

What Is New?

• MPO/calpain activation increases endothelia adhesiveness to leukocytes

• MPO/Calpain activation downregulates AMPK/eNOS signaling

• Nitrosylation of calpain as regulatory mechanism of endothelial the endothelial dysfunction of MPO

• PP2A signals downstream of MPO/Calpain to impair AMPK/eNOS phosphorylation

What Is Relevant?

• MPO is implicated in cardiovascular disease and this study uncovers new molecular targets of MPO in the vascular endothelium.

• The calpain system is amenable to pharmacological inhibition and could offer a viable strategy to avert the damaging effect of MPO in disease states of the cardiovascular system

• Cardiovascular disease is almost invariably associated with insulin resistance and metabolic syndrome. Interestingly, this study demonstrates that MPO downregulates the energy sensing kinase AMPK, a well-established regulator of peripheral insulin action.