Prevention by the CXCR2 antagonist SCH527123 of the calcification of porcine heart valve cusps implanted subcutaneously in rats

IntroductionCalcification is a main cause of bioprosthetic heart valves failure. It may be promoted by the inflammation developed in the glutaraldehyde (GA)-fixed cusps of the bioprosthesis. We tested the hypothesis that antagonizing the C-X-C chemokines receptor 2 (CXCR2) may prevent the calcification of GA-fixed porcine aortic valves.Materiel and methodsFour-week-old Sprague Dawley males were transplanted with 2 aortic valve cusps isolated from independent pigs and implanted into the dorsal wall. Four groups of 6 rats were compared: rats transplanted with GA-free or GA-fixed cusps and rats transplanted with GA-fixed cusps and treated with 1 mg/kg/day SCH5217123 (a CXCR2 antagonist) intraperitoneally (IP) or subcutaneously (SC) around the xenograft, for 14 days. Then, rats underwent blood count before xenografts have been explanted for histology and biochemistry analyses.ResultsA strong calcification of the xenografts was induced by GA pre-incubation. However, we observed a significant decrease in this effect in rats treated with SCH527123 IP or SC. Implantation of GA-fixed cusps was associated with a significant increase in the white blood cell count, an effect that was significantly prevented by SCH527123. In addition, the expression of the CD3, CD68 and CXCR2 markers was reduced in the GA-fixed cusps explanted from rats treated with SCH527123 as compared to those explanted from non-treated rats.ConclusionThe calcification of GA-fixed porcine aortic valve cusps implanted subcutaneously in rats was significantly prevented by antagonizing CXCR2 with SCH527123. This effect may partly result from an inhibition of the GA-induced infiltration of T-cells and macrophages into the xenograft

Rôle de l'interleukine-8 dans la progression de la calcification valvulaire aortique

Calcific aortic valve stenosis (CAVS) is characterized by sclerosis of the valve leaflets associated with calcium deposition. The inability of the valve leaflets to open completely constitutes an obstacle to left ventricular ejection. The resulting functional cardiac impact is then responsible for major morbidity and mortality. The pathophysiological mechanisms are still poorly understood, but low-level chronic inflammation of valve tissue plays a critical role in disease initiation and progression. In this work, we hypothesized that interleukin 8 (IL-8), a pro-inflammatory cytokine which the MP3CV laboratory has already shown to be involved in vascular calcification, also amplifies aortic valve calcification, and that blocking CXCR1/2 receptors could reduce this calcification. To verify this hypothesis, we worked on valve interstitial cells (hVICs) isolated from aortic valves from patients who had undergone surgical valve replacement due to CAVS. We have shown that hVICs express CXCR1 and CXCR2. The addition of IL-8 to a proacifying medium ([Pi] = 3.8 mM) induced within hVICs the activation of the NFkB pathway and this activation was prevented by pharmacologically blocking CXCR1/2 (SCH522123 compound). In the presence of Pi, IL-8 induced a significant increase in the calcification of hVICs. This effect was reduced by blocking CXCR1/2 or inhibiting NFkB. Moreover, when the hVICs were exposed to sera from patients with CAVS, an increase in their calcification was observed and this was prevented by the presence of SCH527123. This procalcifying effect of IL-8 has been associated with an increase in the degradation of elastin and involves the NFkB signaling pathway and the expression of the metalloprotease MMP12. Furthermore, it can be prevented by blocking CXCR1/2 or inhibiting NFKB or MMP12. In this work, we also analyzed the valves of patients with CAVS in immunohistochemistry. A higher expression of CXCR1, CXCR2 and MMP12 in calcified areas compared to non-calcified areas was demonstrated. At the same time, the expression of the intact form of elastin was reduced in the calcified areas, suggesting its degradation, an observation confirmed using Western blot. In addition, aortic valves from healthy pigs were cultured and we were able to confirm in this model the amplifying effect of IL-8 on valve calcification via a mechanism probably dependent on CXCR1/2, NFkB and MMP12. In a second part of the work, we were interested in testing the effect of pharmacological blockade of CXCR1/2 in an animal model of ectopic valve calcification. This model consisted of implanting pig aortic valve leaflets previously exposed to glutaraldehyde (GA) at the dorsal level in rats, via the subcutaneous route, then evaluating the calcification of these tissues after 14 days. GA is a chemical agent frequently used in the preservation and tanning of valve bioprostheses. In this model, we demonstrated a significant preventive effect of intraperitoneal administration of SCH527123 in rats on the calcification of the implanted valve. This effect was associated with a lower expression in implanted valves of the markers CD3, CD11 and CD68, suggesting a reduction in leukocyte infiltration. In conclusion, this work highlighted for the first time a deleterious role of IL-8 in aortic valve calcification and identified new potential therapeutic targets including CXCR1/2, NFkB and MMP12. IL-8 could exert its procalcifying effect directly on hVICs in native valves but also indirectly via its effect on leukocyte chemotaxis, such as in valvular bioprosthesesLe rétrécissement aortique calcifié (RAC) est une pathologie cardiaque grave caractérisée par une sclérose des feuillets valvulaires aortiques associée à des dépôts de calcium. L'incapacité des feuillets valvulaires à s'ouvrir complètement constitue un obstacle à l'éjection ventriculaire gauche. Le retentissement cardiaque fonctionnel qui en résulte est alors responsable d'une morbi-mortalité importante. Les mécanismes physiopathologiques impliqués restent encore mal compris mais l'inflammation chronique joue un rôle critique dans l'initiation et la progression de la maladie. Dans ce travail, nous avons fait l'hypothèse que l'interleukine 8 (IL-8), une cytokine pro-inflammatoire dont le laboratoire MP3CV a déjà montré l'implication dans la calcification vasculaire, amplifie également la calcification de la valve aortique, et que le blocage des récepteurs CXCR1/2 pourrait réduire cette calcification. Pour vérifier cette hypothèse nous avons travaillé sur des cellules interstitielles de valves (hVICs) isolées de valves aortiques issues de patients ayant subi un remplacement valvulaire chirurgical du fait d'un RAC. Nous avons montré que les hVICs expriment CXCR1 et CXCR2. L'ajout d'IL-8 à un milieu procacifiant ([Pi] = 3,8 mM) a induit au sein des hVICs l'activation de la voie NFkB et cette activation a été prévenue en bloquant pharmacologiquement CXCR1/2 (agent SCH522123). En présence de Pi, l'IL-8 a induit une augmentation de la calcification des hVICs. Cet effet a été réduit en bloquant CXCR1/2 ou NFkB. Par ailleurs, lorsque les hVICs ont été exposées aux sérums de patients atteints de RAC, une augmentation de leur calcification a été observée et celle-ci a été prévenue en présence de SCH527123. L'effet procalcifiant de l'IL-8 a été associé à une augmentation de la dégradation de l'élastine et a pu être prévenu en bloquant pharmacologiquement les récepteurs CXCR1/2 de l'IL-8, la voie NFKB ou la MMP12. Dans ce travail, nous avons également analysé les valves de patients atteints de RAC en immunohistologie. Une expression plus importante de CXCR1, CXCR2 et de MMP12 dans les zones calcifiées en comparaison avec les zones non calcifiées a été mise en évidence. Parallèlement, l'expression de la forme intacte d'élastine était réduite dans les zones calcifiées suggérant sa dégradation, observation confirmée par Western blot. Par ailleurs, des valves aortiques de porcs sains ont été mises en culture et nous avons pu confirmer dans ce modèle l'effet amplificateur de l'IL-8 sur la calcification de la valve via un mécanisme dépendant probablement de CXCR1/2, NFkB et MMP12. Dans une seconde partie du travail, nous nous sommes intéressés à tester l'effet du blocage pharmacologique de CXCR1/2 dans un modèle animal de calcification valvulaire ectopique. Ce modèle a consisté à implanter au niveau dorsal chez le rat, par voie sous-cutanée, des feuillets valvulaires aortiques de porc préalablement exposés au glutaraldéhyde (GA) puis à évaluer la calcification de ces tissus après 14 jours. Le GA est un agent chimique utilisé dans la conservation et le tannage des bioprothèses valvulaires. Nous avons observé un effet préventif de l'administration chez le rat de SCH527123 par voie intrapéritonéale sur la calcification de la valve implantée. Cet effet était associé à une moindre expression au niveau des valves implantées des marqueurs CD3, CD11 et CD68, suggérant une réduction de l'infiltration leucocytaire. En conclusion, ce travail a mis en lumière pour la première fois un rôle délétère de l'IL-8 dans la calcification valvulaire aortique et a identifié de nouvelles cibles thérapeutiques potentielles incluant CXCR1/2, NFkB et MMP12. L'IL-8 pourrait exercer son effet procalcifiant de façon directe sur les hVICs dans les valves natives mais également de façon indirecte via son effet sur le chimiotactisme des leucocytes notamment dans les bioprothèses valvulaire

Role of interleukin-8 in the progression of aortic valve calcification

Le rétrécissement aortique calcifié (RAC) est une pathologie cardiaque grave caractérisée par une sclérose des feuillets valvulaires aortiques associée à des dépôts de calcium. L'incapacité des feuillets valvulaires à s'ouvrir complètement constitue un obstacle à l'éjection ventriculaire gauche. Le retentissement cardiaque fonctionnel qui en résulte est alors responsable d'une morbi-mortalité importante. Les mécanismes physiopathologiques impliqués restent encore mal compris mais l'inflammation chronique joue un rôle critique dans l'initiation et la progression de la maladie. Dans ce travail, nous avons fait l'hypothèse que l'interleukine 8 (IL-8), une cytokine pro-inflammatoire dont le laboratoire MP3CV a déjà montré l'implication dans la calcification vasculaire, amplifie également la calcification de la valve aortique, et que le blocage des récepteurs CXCR1/2 pourrait réduire cette calcification. Pour vérifier cette hypothèse nous avons travaillé sur des cellules interstitielles de valves (hVICs) isolées de valves aortiques issues de patients ayant subi un remplacement valvulaire chirurgical du fait d'un RAC. Nous avons montré que les hVICs expriment CXCR1 et CXCR2. L'ajout d'IL-8 à un milieu procacifiant ([Pi] = 3,8 mM) a induit au sein des hVICs l'activation de la voie NFkB et cette activation a été prévenue en bloquant pharmacologiquement CXCR1/2 (agent SCH522123). En présence de Pi, l'IL-8 a induit une augmentation de la calcification des hVICs. Cet effet a été réduit en bloquant CXCR1/2 ou NFkB. Par ailleurs, lorsque les hVICs ont été exposées aux sérums de patients atteints de RAC, une augmentation de leur calcification a été observée et celle-ci a été prévenue en présence de SCH527123. L'effet procalcifiant de l'IL-8 a été associé à une augmentation de la dégradation de l'élastine et a pu être prévenu en bloquant pharmacologiquement les récepteurs CXCR1/2 de l'IL-8, la voie NFKB ou la MMP12. Dans ce travail, nous avons également analysé les valves de patients atteints de RAC en immunohistologie. Une expression plus importante de CXCR1, CXCR2 et de MMP12 dans les zones calcifiées en comparaison avec les zones non calcifiées a été mise en évidence. Parallèlement, l'expression de la forme intacte d'élastine était réduite dans les zones calcifiées suggérant sa dégradation, observation confirmée par Western blot. Par ailleurs, des valves aortiques de porcs sains ont été mises en culture et nous avons pu confirmer dans ce modèle l'effet amplificateur de l'IL-8 sur la calcification de la valve via un mécanisme dépendant probablement de CXCR1/2, NFkB et MMP12. Dans une seconde partie du travail, nous nous sommes intéressés à tester l'effet du blocage pharmacologique de CXCR1/2 dans un modèle animal de calcification valvulaire ectopique. Ce modèle a consisté à implanter au niveau dorsal chez le rat, par voie sous-cutanée, des feuillets valvulaires aortiques de porc préalablement exposés au glutaraldéhyde (GA) puis à évaluer la calcification de ces tissus après 14 jours. Le GA est un agent chimique utilisé dans la conservation et le tannage des bioprothèses valvulaires. Nous avons observé un effet préventif de l'administration chez le rat de SCH527123 par voie intrapéritonéale sur la calcification de la valve implantée. Cet effet était associé à une moindre expression au niveau des valves implantées des marqueurs CD3, CD11 et CD68, suggérant une réduction de l'infiltration leucocytaire. En conclusion, ce travail a mis en lumière pour la première fois un rôle délétère de l'IL-8 dans la calcification valvulaire aortique et a identifié de nouvelles cibles thérapeutiques potentielles incluant CXCR1/2, NFkB et MMP12. L'IL-8 pourrait exercer son effet procalcifiant de façon directe sur les hVICs dans les valves natives mais également de façon indirecte via son effet sur le chimiotactisme des leucocytes notamment dans les bioprothèses valvulairesCalcific aortic valve stenosis (CAVS) is characterized by sclerosis of the valve leaflets associated with calcium deposition. The inability of the valve leaflets to open completely constitutes an obstacle to left ventricular ejection. The resulting functional cardiac impact is then responsible for major morbidity and mortality. The pathophysiological mechanisms are still poorly understood, but low-level chronic inflammation of valve tissue plays a critical role in disease initiation and progression. In this work, we hypothesized that interleukin 8 (IL-8), a pro-inflammatory cytokine which the MP3CV laboratory has already shown to be involved in vascular calcification, also amplifies aortic valve calcification, and that blocking CXCR1/2 receptors could reduce this calcification. To verify this hypothesis, we worked on valve interstitial cells (hVICs) isolated from aortic valves from patients who had undergone surgical valve replacement due to CAVS. We have shown that hVICs express CXCR1 and CXCR2. The addition of IL-8 to a proacifying medium ([Pi] = 3.8 mM) induced within hVICs the activation of the NFkB pathway and this activation was prevented by pharmacologically blocking CXCR1/2 (SCH522123 compound). In the presence of Pi, IL-8 induced a significant increase in the calcification of hVICs. This effect was reduced by blocking CXCR1/2 or inhibiting NFkB. Moreover, when the hVICs were exposed to sera from patients with CAVS, an increase in their calcification was observed and this was prevented by the presence of SCH527123. This procalcifying effect of IL-8 has been associated with an increase in the degradation of elastin and involves the NFkB signaling pathway and the expression of the metalloprotease MMP12. Furthermore, it can be prevented by blocking CXCR1/2 or inhibiting NFKB or MMP12. In this work, we also analyzed the valves of patients with CAVS in immunohistochemistry. A higher expression of CXCR1, CXCR2 and MMP12 in calcified areas compared to non-calcified areas was demonstrated. At the same time, the expression of the intact form of elastin was reduced in the calcified areas, suggesting its degradation, an observation confirmed using Western blot. In addition, aortic valves from healthy pigs were cultured and we were able to confirm in this model the amplifying effect of IL-8 on valve calcification via a mechanism probably dependent on CXCR1/2, NFkB and MMP12. In a second part of the work, we were interested in testing the effect of pharmacological blockade of CXCR1/2 in an animal model of ectopic valve calcification. This model consisted of implanting pig aortic valve leaflets previously exposed to glutaraldehyde (GA) at the dorsal level in rats, via the subcutaneous route, then evaluating the calcification of these tissues after 14 days. GA is a chemical agent frequently used in the preservation and tanning of valve bioprostheses. In this model, we demonstrated a significant preventive effect of intraperitoneal administration of SCH527123 in rats on the calcification of the implanted valve. This effect was associated with a lower expression in implanted valves of the markers CD3, CD11 and CD68, suggesting a reduction in leukocyte infiltration. In conclusion, this work highlighted for the first time a deleterious role of IL-8 in aortic valve calcification and identified new potential therapeutic targets including CXCR1/2, NFkB and MMP12. IL-8 could exert its procalcifying effect directly on hVICs in native valves but also indirectly via its effect on leukocyte chemotaxis, such as in valvular bioprosthese

Prevention by the CXCR2 antagonist SCH527123 of the calcification of porcine heart valve cusps implanted subcutaneously in rats

International audienceIntroduction Calcification is a main cause of bioprosthetic heart valves failure. It may be promoted by the inflammation developed in the glutaraldehyde (GA)-fixed cusps of the bioprosthesis. We tested the hypothesis that antagonizing the C-X-C chemokines receptor 2 (CXCR2) may prevent the calcification of GA-fixed porcine aortic valves. Materiel and methods Four-week-old Sprague Dawley males were transplanted with 2 aortic valve cusps isolated from independent pigs and implanted into the dorsal wall. Four groups of 6 rats were compared: rats transplanted with GA-free or GA-fixed cusps and rats transplanted with GA-fixed cusps and treated with 1 mg/kg/day SCH5217123 (a CXCR2 antagonist) intraperitoneally (IP) or subcutaneously (SC) around the xenograft, for 14 days. Then, rats underwent blood count before xenografts have been explanted for histology and biochemistry analyses. Results A strong calcification of the xenografts was induced by GA pre-incubation. However, we observed a significant decrease in this effect in rats treated with SCH527123 IP or SC. Implantation of GA-fixed cusps was associated with a significant increase in the white blood cell count, an effect that was significantly prevented by SCH527123. In addition, the expression of the CD3, CD68 and CXCR2 markers was reduced in the GA-fixed cusps explanted from rats treated with SCH527123 as compared to those explanted from non-treated rats. Conclusion The calcification of GA-fixed porcine aortic valve cusps implanted subcutaneously in rats was significantly prevented by antagonizing CXCR2 with SCH527123. This effect may partly result from an inhibition of the GA-induced infiltration of T-cells and macrophages into the xenograft

Datasheet1_Prevention by the CXCR2 antagonist SCH527123 of the calcification of porcine heart valve cusps implanted subcutaneously in rats.docx

IntroductionCalcification is a main cause of bioprosthetic heart valves failure. It may be promoted by the inflammation developed in the glutaraldehyde (GA)-fixed cusps of the bioprosthesis. We tested the hypothesis that antagonizing the C-X-C chemokines receptor 2 (CXCR2) may prevent the calcification of GA-fixed porcine aortic valves.Materiel and methodsFour-week-old Sprague Dawley males were transplanted with 2 aortic valve cusps isolated from independent pigs and implanted into the dorsal wall. Four groups of 6 rats were compared: rats transplanted with GA-free or GA-fixed cusps and rats transplanted with GA-fixed cusps and treated with 1 mg/kg/day SCH5217123 (a CXCR2 antagonist) intraperitoneally (IP) or subcutaneously (SC) around the xenograft, for 14 days. Then, rats underwent blood count before xenografts have been explanted for histology and biochemistry analyses.ResultsA strong calcification of the xenografts was induced by GA pre-incubation. However, we observed a significant decrease in this effect in rats treated with SCH527123 IP or SC. Implantation of GA-fixed cusps was associated with a significant increase in the white blood cell count, an effect that was significantly prevented by SCH527123. In addition, the expression of the CD3, CD68 and CXCR2 markers was reduced in the GA-fixed cusps explanted from rats treated with SCH527123 as compared to those explanted from non-treated rats.ConclusionThe calcification of GA-fixed porcine aortic valve cusps implanted subcutaneously in rats was significantly prevented by antagonizing CXCR2 with SCH527123. This effect may partly result from an inhibition of the GA-induced infiltration of T-cells and macrophages into the xenograft.</p

Combination of Angiotensin (1-7) Agonists and Convalescent Plasma as a New Strategy to Overcome Angiotensin Converting Enzyme 2 (ACE2) Inhibition for the Treatment of COVID-19

Coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently the most concerning health problem worldwide. SARS-CoV-2 infects cells by binding to angiotensin-converting enzyme 2 (ACE2). It is believed that the differential response to SARS-CoV-2 is correlated with the differential expression of ACE2. Several reports proposed the use of ACE2 pharmacological inhibitors and ACE2 antibodies to block viral entry. However, ACE2 inhibition is associated with lung and cardiovascular pathology and would probably increase the pathogenesis of COVID-19. Therefore, utilizing ACE2 soluble analogs to block viral entry while rescuing ACE2 activity has been proposed. Despite their protective effects, such analogs can form a circulating reservoir of the virus, thus accelerating its spread in the body. Levels of ACE2 are reduced following viral infection, possibly due to increased viral entry and lysis of ACE2 positive cells. Downregulation of ACE2/Ang (1-7) axis is associated with Ang II upregulation. Of note, while Ang (1-7) exerts protective effects on the lung and cardiovasculature, Ang II elicits pro-inflammatory and pro-fibrotic detrimental effects by binding to the angiotensin type 1 receptor (AT1R). Indeed, AT1R blockers (ARBs) can alleviate the harmful effects associated with Ang II upregulation while increasing ACE2 expression and thus the risk of viral infection. Therefore, Ang (1-7) agonists seem to be a better treatment option. Another approach is the transfusion of convalescent plasma from recovered patients with deteriorated symptoms. Indeed, this appears to be promising due to the neutralizing capacity of anti-COVID-19 antibodies. In light of these considerations, we encourage the adoption of Ang (1-7) agonists and convalescent plasma conjugated therapy for the treatment of COVID-19 patients. This therapeutic regimen is expected to be a safer choice since it possesses the proven ability to neutralize the virus while ensuring lung and cardiovascular protection through modulation of the inflammatory response

Aortic valve calcification is promoted by interleukin-8 and restricted through antagonizing CXCR2

International audienceAims Inflammatory cytokines play a critical role in the progression of calcific aortic valve disease (CAVD), for which there is currently no pharmacological treatment. The aim of this study was to test the hypothesis that interleukin-8 (IL-8), known to be involved in arterial calcification, also promotes aortic valve calcification (AVC) and to evaluate whether pharmacologically blocking the IL-8 receptor, CXC motif chemokine receptor 2 (CXCR2), could be effective in preventing AVC progression. Methods and Results A cohort of 195 patients (median age 73, 74% men) diagnosed with aortic valve stenosis (severe in 16.9% of cases) were prospectively followed by CT for a median time of 2.6 years. A Cox proportional hazards regression analysis indicated that baseline IL-8 serum concentrations were associated with rapid progression of AVC, defined as an annualized change in the calcification score by CT ≥ 110 AU/year, after adjustment for age, gender, bicuspid anatomy and baseline disease severity. In vitro, exposure of primary human aortic valvular interstitial cells (hVICs) to 15 pg/ml IL-8 induced a two-fold increase in inorganic phosphate (Pi)-induced calcification. IL-8 promoted NFκB pathway activation, MMP-12 expression, and elastin degradation in hVICs exposed to Pi. These effects were prevented by SCH527123, an antagonist of CXCR2. The expression of CXCR2 was confirmed in hVICs and samples of aortic valves isolated from patients with CAVD, in which the receptor was mainly found in calcified areas, along with MMP-12 and a degraded form of elastin. Finally, in a rat model of chronic kidney disease-associated CAVD, SCH527123 treatment (1 mg/kg/day given orally for 11 weeks) limited the decrease in aortic cusp separation, the increase in maximal velocity of the transaortic jet, and the increase in aortic mean pressure gradient measured by echocardiography, effects that were associated with a reduction in hydroxyapatite deposition and MMP-12 expression in the aortic valves. Conclusion Overall, these results highlight, for the first time, a significant role for IL-8 in the progression of CAVD by promoting calcification via a CXCR2­- and MMP-12-dependent mechanism that leads to elastin degradation, and identify CXCR2 as a promising therapeutic target for the treatment of CAVD