A case with familial hypercholesterolemia complicated with severe systemic atherosclerosis intensively treated for more than 30 years.

We present a case of a Japanese patient with familial hypercholesterolemia (FH) caused by a low-density lipoprotein (LDL) receptor gene mutation. A 47-year-old female was referred to our hospital due to her systemic xanthomatosis associated with elevated LDL-cholesterolemia (292 mg/dl). She was diagnosed with heterozygous FH, and started to be treated with simvastatin 10 mg. During her clinical course, she underwent percutaneous coronary intervention (PCI) (at 69 years), coronary artery bypass grafting (CABG) twice (at 62 years, and 75 years), femoral popliteal bypass surgery (at 67 years), together with intensification of lipid-lowering therapies, including proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor. She was admitted to our hospital due to dyspnea on effort, caused by severe aortic valve stenosis as well as sick sinus syndrome at the age of 78 years. transcatheter aortic valve implantation (TAVI) using balloon expandable valve was successfully performed after DDD pacemaker implantation. She was discharged from our hospital without any symptoms. During more than 30 years of treatment period in our institute, we have introduced the latest therapeutic strategies, and treated her intensively. We are proud that we can save life even in this severe case through multiple strategies developed over the decades; however, this case clearly suggests that lipid-lowering therapies should be started much earlier in patients with FH. .

Pharmacological Inhibition of MALT1 Protease Leads to a Progressive IPEX-Like Pathology.

Genetic disruption or short-term pharmacological inhibition of MALT1 protease is effective in several preclinical models of autoimmunity and B cell malignancies. Despite these protective effects, the severe reduction in regulatory T cells (Tregs) and the associated IPEX-like pathology occurring upon congenital disruption of the MALT1 protease in mice has raised concerns about the long-term safety of MALT1 inhibition. Here we describe the results of a series of toxicology studies in rat and dog species using MLT-943, a novel potent and selective MALT1 protease inhibitor. While MLT-943 effectively prevented T cell-dependent B cell immune responses and reduced joint inflammation in the collagen-induced arthritis rat pharmacology model, in both preclinical species, pharmacological inhibition of MALT1 was associated with a rapid and dose-dependent reduction in Tregs and resulted in the progressive appearance of immune abnormalities and clinical signs of an IPEX-like pathology. At the 13-week time point, rats displayed severe intestinal inflammation associated with mast cell activation, high serum IgE levels, systemic T cell activation and mononuclear cell infiltration in multiple tissues. Importantly, using thymectomized rats we demonstrated that MALT1 protease inhibition affects peripheral Treg frequency independently of effects on thymic Treg output and development. Our data confirm the therapeutic potential of MALT1 protease inhibitors but highlight the safety risks and challenges to consider before potential application of such inhibitors into the clinic.

Malt1 Protease Deficiency in Mice Disrupts Immune Homeostasis at Environmental Barriers and Drives Systemic T Cell-Mediated Autoimmunity.

The paracaspase Malt1 is a key regulator of canonical NF-κB activation downstream of multiple receptors in both immune and nonimmune cells. Genetic disruption of Malt1 protease function in mice and MALT1 mutations in humans results in reduced regulatory T cells and a progressive multiorgan inflammatory pathology. In this study, we evaluated the altered immune homeostasis and autoimmune disease in Malt1 protease-deficient (Malt1PD) mice and the Ags driving disease manifestations. Our data indicate that B cell activation and IgG1/IgE production is triggered by microbial and dietary Ags preferentially in lymphoid organs draining mucosal barriers, likely as a result of dysregulated mucosal immune homeostasis. Conversely, the disease was driven by a polyclonal T cell population directed against self-antigens. Characterization of the Malt1PD T cell compartment revealed expansion of T effector memory cells and concomitant loss of a CD4+ T cell population that phenotypically resembles anergic T cells. Therefore, we propose that the compromised regulatory T cell compartment in Malt1PD animals prevents the efficient maintenance of anergy and supports the progressive expansion of pathogenic, IFN-γ-producing T cells. Overall, our data revealed a crucial role of the Malt1 protease for the maintenance of intestinal and systemic immune homeostasis, which might provide insights into the mechanisms underlying IPEX-related diseases associated with mutations in MALT1.

Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity.

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells downstream of pattern recognition receptors. Best studied are the Dectin-1 and Dectin-2 members of the C-type lectin-like receptor family that induce a SYK- and CARD9-dependent signaling cascade leading to NF-κB activation, in a MALT1-dependent manner. By contrast, Toll-like receptors (TLR), such as TLR-4, propagate NF-κB activation but signal via an MYD88/IRAK-dependent cascade. Nonetheless, whether MALT1 might contribute to TLR-4 signaling has remained unclear. Recent evidence with MLT-827, a potent and selective inhibitor of MALT1 paracaspase activity, indicates that TNF- production downstream of TLR-4 in human myeloid cells is independent of MALT1, as opposed to TNF- production downstream of Dectin-1, which is MALT1 dependent. Here, we addressed the selective involvement of MALT1 in pattern recognition sensing further, using a variety of human and mouse cellular preparations, and stimulation of Dectin-1, MINCLE or TLR-4 pathways. We also provided additional insights by exploring cytokines beyond TNF-, and by comparing MLT-827 to a SYK inhibitor (Cpd11) and to an IKK inhibitor (AFN700). Collectively, the data provided further evidence for the MALT1-dependency of C-type lectin-like receptor -signaling by contrast to TLR-signaling.

Deficiency of MALT1 paracaspase activity results in unbalanced regulatory and effector T and B cell responses leading to multiorgan inflammation.

The paracaspase MALT1 plays an important role in immune receptor-driven signaling pathways leading to NF-κB activation. MALT1 promotes signaling by acting as a scaffold, recruiting downstream signaling proteins, as well as by proteolytic cleavage of multiple substrates. However, the relative contributions of these two different activities to T and B cell function are not well understood. To investigate how MALT1 proteolytic activity contributes to overall immune cell regulation, we generated MALT1 protease-deficient mice (Malt1(PD/PD)) and compared their phenotype with that of MALT1 knockout animals (Malt1(-/-)). Malt1(PD/PD) mice displayed defects in multiple cell types including marginal zone B cells, B1 B cells, IL-10-producing B cells, regulatory T cells, and mature T and B cells. In general, immune defects were more pronounced in Malt1(-/-) animals. Both mouse lines showed abrogated B cell responses upon immunization with T-dependent and T-independent Ags. In vitro, inactivation of MALT1 protease activity caused reduced stimulation-induced T cell proliferation, impaired IL-2 and TNF-α production, as well as defective Th17 differentiation. Consequently, Malt1(PD/PD) mice were protected in a Th17-dependent experimental autoimmune encephalomyelitis model. Surprisingly, Malt1(PD/PD) animals developed a multiorgan inflammatory pathology, characterized by Th1 and Th2/0 responses and enhanced IgG1 and IgE levels, which was delayed by wild-type regulatory T cell reconstitution. We therefore propose that the pathology characterizing Malt1(PD/PD) animals arises from an immune imbalance featuring pathogenic Th1- and Th2/0-skewed effector responses and reduced immunosuppressive compartments. These data uncover a previously unappreciated key function of MALT1 protease activity in immune homeostasis and underline its relevance in human health and disease.

Hurdles in the drug discovery of cathepsin K inhibitors.

There were many hurdles in the drug discovery of cathepsin K inhibitors such as species differences not only in bone metabolism but also in amino acid sequences in the critical site of the target enzyme, discrepancies between PK/PD due to unique tissue distribution of the inhibitor affecting both efficacy and side effects originated from a characteristic intracellular or tissue distribution of some classes of compounds. The value of this new therapeutic approach over the launched indirect competitors should be further clarified from the efficacy and side effect point of view. The cathepsin K inhibitor drug discovery was initiated based on a strong and osteoclast-specific expression of this enzyme. However, the tissues and cells expressing cathepsin K have been expanding as the investigation on pathological conditions progressed with respect to side effects as well as new possible indications.

Ovalbumin-induced plasma interleukin-4 levels are reduced in ceramide kinase-deficient DO11.10 RAG1-/- mice.

Ceramide kinase (CERK) produces the bioactive lipid ceramide-1-phosphate (C1P) and is a key regulator of ceramide and dihydroceramide levels. It is likely that CERK and C1P play a role in inflammatory processes but the cells involved and the mechanisms used remain to be clarified. In particular, the impact of CERK on T-cell biology has not been studied so far. Here, we used Cerk-/- mice backcrossed with DO11.10/RAG1-/- mice to probe the effect of CERK ablation on T-cell activation. Levels of interleukin (IL)-2, IL-4, IL-5, IL-13, of tumor necrosis factor (TNF)-alpha, and of interferon (INF)-gamma were recorded following ovalbumin challenge in vivo and using ovalbumin-treated splenocytes ex- vivo. Absence of CERK led to a significant decrease in the production of IL-4, thus suggesting that CERK may polarize T cells towards the TH2 cell subtype. However, the importance of CERK to TH2 cell biology will have to be investigated further because in a model of asthma, which is TH2-cell driven, Cerk-/- mice responded like wild-type animals.

Ceramide kinase deficiency impairs microendothelial cell angiogenesis in vitro.

The recent generation of ceramide kinase (CerK)-deficient (Cerk (-/-)) mice as well as the identification of the potent CerK inhibitor NVP-231 have provided unprecedented opportunities to better understand CerK biology. Here we used skin dermal microendothelial cells (DMECs) and we show that CerK activity regulates their neovascularization in a matrigel environment in vitro. Capillary-like tube formation was significantly impaired in CerK-deficient cells or in wild-type (WT) cells treated with NVP-231 as compared with untreated WT cells. This was not the result of compromised proliferation or survival because Cerk (-/-) endothelial cells were able to migrate out of dermal fragments and grow in monolayer culture as well as their WT counterpart. Vascular endothelial growth factor, fibroblast growth factor or tumor necrosis factor could not rescue the angiogenesis defect observed in Cerk (-/-) DMEMs. Moreover, CerK ablation increased serum ceramide levels at the expense of dihydroceramide levels without affecting sphingosine, dihydrosphingosine, sphingosine-1-phosphate or dihydrosphingosine-1-phosphate levels. These observations collectively suggest that CerK-catalyzed formation of C1P may regulate angiogenesis by a novel mechanism that is independent of S1P formation and signaling.