S100A4-neutralizing monoclonal antibody 6B12 counteracts the established experimental skin fibrosis induced by bleomycin.

OBJECTIVES: Our previous studies have demonstrated that the Damage Associated Molecular Pattern (DAMP) protein, S100A4, is overexpressed in the involved skin and peripheral blood of patients with SSc. It is associated with skin and lung involvement, and disease activity. By contrast, lack of S100A4 prevented the development of experimental dermal fibrosis. Herein we aimed to evaluate the effect of murine anti-S100A4 mAb 6B12 in the treatment of preestablished experimental dermal fibrosis. METHODS: The effects of 6B12 were assessed at therapeutic dosages in a modified bleomycin-induced dermal fibrosis mouse model by evaluating fibrotic (dermal thickness, proliferation of myofibroblasts, hydroxyproline content, phosphorylated Smad3-positive cell count) and inflammatory (leukocytes infiltrating the lesional skin, systemic levels of selected cytokines and chemokines) outcomes, and transcriptional profiling (RNA sequencing). RESULTS: Treatment with 7.5 mg/kg 6B12 attenuated and might even reduce pre-existing dermal fibrosis induced by bleomycin as evidenced by reduction in dermal thickness, myofibroblast count and collagen content. These antifibrotic effects were mediated by the downregulation of TGF-ß/Smad signalling and partially by reducing the number of leukocytes infiltrating the lesional skin and decrease in the systemic levels of IL-1α, eotaxin, CCL2 and CCL5. Moreover, transcriptional profiling demonstrated that 7.5 mg/kg 6B12 also modulated several profibrotic and proinflammatory processes relevant to the pathogenesis of SSc. CONCLUSION: Targeting S100A4 by the 6B12 mAb demonstrated potent antifibrotic and anti-inflammatory effects on bleomycin-induced dermal fibrosis and provided further evidence for the vital role of S100A4 in the pathophysiology of SSc.

Clinical and pathogenic significance of S100A4 overexpression in systemic sclerosis.

OBJECTIVES: We have studied the damage-associated molecular pattern protein S100A4 as a driver of fibroblast activation in systemic sclerosis (SSc). METHODS: S100A4 protein concentration was measured by ELISA in serum of SSc (n=94) and healthy controls (n=15). Protein expression in skin fibroblast cultures from diffuse cutaneous SSc (SScF, n=6) and healthy controls (normal fibroblasts (NF), n=6) was assessed. Recombinant S100A4 and a high affinity anti-S100A4 neutralising monoclonal antibody (AX-202) were tested on SScF and NF. RESULTS: Median (range) S100A4 (ng/mL) was higher in serum of SSc (89.9 (15.0-240.0)) than healthy controls (71.4 (7.9-131.8); p=0.027). There was association with SSc-interstitial lung disease (p=0.025, n=55), scleroderma renal crisis (p=0.026, n=4). Median (range) S100A4 (ng/mL) was higher in culture supernatants of SScF (4.19 (0.52-8.42)) than NF controls (0.28 (0.02-3.29); p<0.0001). AX-202 reduced the constitutive profibrotic gene and protein expression phenotype of SScF. Genome-wide RNA sequencing analysis identified an S100A4 activated signature in NF overlapping the hallmark gene expression signature of SScF. Thus, 464 differentially expressed genes (false discovery rate (FDR) <0.001 and fold change (FC) >1.5) induced in NF by S100A4 were also constitutively overexpressed, and downregulated by AX-202, in SScF. Pathway mapping of these S100A4 dependent genes in SSc showed the most significant enriched Kegg pathways (FDR <0.001) were regulation of stem cell pluripotency (4.6-fold) and metabolic pathways (1.9-fold). CONCLUSION: Our findings provide compelling evidence for a profibrotic role for S100A4 in SSc and suggest that serum level may be a biomarker of major organ manifestations and disease severity. This study supports examining the therapeutic potential of targeting S100A4 in SSc.

CaMKII in addition to MLCK contributes to phosphorylation of regulatory light chain in cardiomyocytes.

The aim was to identify kinase activities involved in the phosphorylation of regulatory light chain (RLC) in situ in cardiomyocytes. In electrically stimulated rat cardiomyocytes, phosphatase inhibition by calyculin A unmasked kinase activities evoking an increase of phosphorylated RLC (P-RLC) from about 16% to about 80% after 80 min. The phosphorylation rate in cardiomyocytes was reduced by about 40% by the myosin light chain kinase (MLCK) inhibitor, ML-7. In rat ventricular muscle strips, calyculin A induced a positive inotropic effect that correlated with P-RLC levels. The inotropic effect and P-RLC elevation were abolished by ML-7 treatment. The kinase activities phosphorylating RLC in cardiomyocytes were reduced by about 60% by the non-selective kinase inhibitor staurosporine and by about 50% by the calmodulin antagonist W7. W7 eliminated the inhibitory effect of ML-7, suggesting that the cardiac MLCK is Ca(2+)/calmodulin (CaM)-dependent. The CaM-dependent kinase II (CaMKII) inhibitor KN-93 attenuated the calyculin A-induced RLC phosphorylation by about 40%, indicating a contribution from CaMKII. The residual phosphorylation in the presence of W7 indicated that also CaM-independent kinase activities might contribute. RLC phosphorylation was insensitive to protein kinase C inhibition. In conclusion, in addition to MLCK, CaMKII phosphorylates RLC in cardiomyocytes. Involvement of other kinases cannot be excluded.

Effect of Anti-S100A4 Monoclonal Antibody Treatment on Experimental Skin Fibrosis and Systemic Sclerosis-Specific Transcriptional Signatures in Human Skin.

OBJECTIVE: S100A4 is a DAMP protein. S100A4 is overexpressed in patients with systemic sclerosis (SSc), and levels correlate with organ involvement and disease activity. S100A4-/- mice are protected from fibrosis. The aim of this study was to assess the antifibrotic effects of anti-S100A4 monoclonal antibody (mAb) in murine models of SSc and in precision cut skin slices of patients with SSc. METHODS: The effects of anti-S100A4 mAbs were evaluated in a bleomycin-induced skin fibrosis model and in Tsk-1 mice with a therapeutic dosing regimen. In addition, the effects of anti-S100A4 mAbs on precision cut SSc skin slices were analyzed by RNA sequencing. RESULTS: Inhibition of S100A4 was effective in the treatment of pre-established bleomycin-induced skin fibrosis and in regression of pre-established fibrosis with reduced dermal thickening, myofibroblast counts, and collagen accumulation. Transcriptional profiling demonstrated targeting of multiple profibrotic and proinflammatory processes relevant to the pathogenesis of SSc on targeted S100A4 inhibition in a bleomycin-induced skin fibrosis model. Moreover, targeted S100A4 inhibition also modulated inflammation- and fibrosis-relevant gene sets in precision cut SSc skin slices in an ex vivo trial approach. Selected downstream targets of S100A4, such as AMP-activated protein kinase, calsequestrin-1, and phosphorylated STAT3, were validated on the protein level, and STAT3 inhibition was shown to prevent the profibrotic effects of S100A4 on fibroblasts in human skin. CONCLUSION: Inhibition of S100A4 confers dual targeting of inflammatory and fibrotic pathways in complementary mouse models of fibrosis and in SSc skin. These effects support the further development of anti-S100A4 mAbs as disease-modifying targeted therapies for SSc.

N,N'-Bis(4-hy-droxy-phen-yl)pyridine-2,6-dicarboxamide di-methyl-formamide monosolvate.

The mol-ecular structure of the pyridine derivative, C19H15N3O4·C3H7NO, shows almost planar geometry with dihedral angles of 6.9 (1) and 13.4 (1)° between the pyridine ring and the two benzene rings. This conformation is stabilized by two intra-molecular N-H⋯N(pyridine) bonds. In the crystal, strong O-H⋯O(carboxamide) and N-H⋯O(hy-droxy-phen-yl) hydrogen bonds link the mol-ecules, forming a three-dimensional structure. The di-methyl-formamide solvent mol-ecules are not involved in the hydrogen bonding. The structure shows pseudosymmetry, but refinement in the space group Pbcn leads to significantly worse results and a disordered di-methyl-formamide mol-ecule.

Triethyl-ammonium 4-(3,5-dinitro-benzamido)-N-(3,5-dinitro-benzo-yl)benzene-sulfonamidate.

The mol-ecular structure of the title salt, C6H16N(+)·C20H11N6O12S(-), shows a planar geometry of the benzamido-phen-yl-sulfonyl moiety, with a dihedral angle of 1.59 (9)° between the aromatic ring planes. The central ring and the aromatic ring of the other dinitro-benzamide group are nearly perpendicular, making a dihedral angle of 89.55 (9)°. All nitro groups lie almost in plane with the associated aromatic rings, the O-N-C-C torsion angles ranging from 9.2 (2) to 24.3 (2)°. In the crystal, strong anion-anion N-H⋯O and anion-cation hydrogen bonds form inversion dimers stacked along the a axis. Less prominent anion-anion C-H⋯O inter-actions lead to the formation of a three-dimensional network including anion-anion dimers as well as anion-anion chains along [100?].

N,N'-Bis(4-bromo-phen-yl)pyridine-2,6-dicarboxamide.

The mol-ecule of the title compound, C19H13Br2N3O2, lies about a twofold rotation axis. The benzene ring makes dihedral angles of 8.9 (2) and 16.4 (2)° with the central pyridine ring and the second benzene ring, respectively. An intra-molecular N-H⋯N contact occurs. In the crystal, mol-ecules are connected by pairs of N-H⋯O hydrogen bonds into chains along the c axis.

Ethyl 2-(3,5-dinitro-benzamido)-benzoate.

The title mol-ecule, C(16)H(13)N(3)O(7), is slightly twisted, with the dihedral angle between the two benzene ring planes being 17.4 (1)°. An intra-molecular N-H⋯O hydrogen bond is observed. In the crystal, weak C-H⋯O hydrogen bonds link the mol-ecules into chains along the b axis.

N-(4-Bromo-phen-yl)-3,5-dinitro-benzamide.

The title mol-ecule, C(13)H(8)BrN(3)O(5), is slightly twisted, with the dihedral angle between the two benzene rings being 5.9 (1)°. In the crystal, N-H⋯O hydrogen bonds link the mol-ecules into one-dimensional chains running along [101]. Further stabilization of the crystal structure is provided by π-π inter-actions [shortest centroid-centroid distance = 3.6467 (17) Å].

N-[4-(4-Nitro-phen-oxy)phen-yl]penta-n-amide.

The asymmetric unit of the title compound, C(17)H(18)N(2)O(4), contains two independent mol-ecules (A and B) differing principally in the conformations of the alkyl chains, anti for molecule A and gauche for molecule B. The dihedral angles between the aromatic rings are 82.51 (6) and 82.25 (6)° in the two molecules. In the crystal, amide-amide inter-actions (as N-H⋯O=C) results in distinct chains of A and B mol-ecules running parallel to the a-axis direction. C-H⋯O inter-actions also occur.

Dicyclo-hexyl-ammonium 3,5-dinitro-benzoate.

The asymmetric unit of the title salt, C(12)H(24)N(+)·C(7)H(3)N(2)O(6) (-), contains two cations and two anions. In the crystal, the cations and anions are connected by N-H⋯O hydrogen bonds, forming a 12-membered ring with an R(4) (4)(12) graph-set motif. The center of this 12-membered ring coincides with an inversion centre. π-π stacking is observed between parallel benzene rings [centroid-centriod distance = 3.771 (2) Å].

N,N-Dicyclo-hexyl-4-nitro-benzamide.

The title compound, C(19)H(26)N(2)O(3), crystallizes with two independent mol-ecules in the asymmetric unit which differ in the twist of the phenyl rings with respect to the plane of the amide group [the C-C-C-O torsion angles are 121.5 (3) and -119.6 (3)° in the two mol-ecules. Both cyclo-hexane rings adopt chair conformations. In the crystal, weak C-H⋯O inter-actions occur. The crystal studied was a non-merohedral twin with a minor component of 4.8 (1)%.

N-(4-Chloro-phen-yl)-4-nitro-benzamide.

The title compound, C(13)H(9)ClN(2)O(3), is almost planar, showing a dihedral angle of 4.63 (6)° between the aromatic ring planes. The nitro group also lies in the plane, the C-C-N-O torsion angle being 6.7 (2)°. There is an intamolecular C-H⋯O hydrogen bond. The crystal structure features N-H⋯O(nitro) hydrogen bonds that link the mol-ecules into zigzag chains extending along [010].

N,N-Dicyclo-hexyl-cyclo-hexa-ne-carboxamide.

In the title compound, C(19)H(33)NO, all three cyclo-hexane rings adopt chair conformations. The crystal packing features weak C-H⋯O inter-actions, forming a supra-molecular chain along the c axis.

A co-crystal of 3-(3,5-dinitro-benzo-yl)-1,1-dimethyl-thio-urea and N,N-dimethyl-3,5-dinitro-benzamide.

In the title compound, C(10)H(10)N(4)O(5)S·C(9)H(9)N(3)O(5), the amide groups of 3-(3,5-dinitro-benzo-yl)-1,1-dimethyl-thio-urea and N,N-dimethyl-3,5-dinitro-benzamide mol-ecules are oriented at dihedral angles of 39.13 (8) and 55.97 (11)°, respectively, to the attached benzene rings. In the crystal, the two mol-ecules are linked by an N-H⋯O hydrogen bond. Weak C-H⋯O link the mol-ecules into a sheet parallel to the bc plane. C-H⋯S inter-actions also occur.

N,N-Dicyclo-hexyl-3,5-dinitro-benzamide.

In the title compound, C(19)H(25)N(3)O(5), the benzene ring is not coplanar with the amide group [dihedral angle = 61.90 (5)°]. The cyclo-hexyl rings are in chair conformations. There is a strong inter-molecular inter-action between the C=O group of the amide group and the nitro group of an adjoining mol-ecule, with a short O⋯N distance of 2.7862 (17) Å. In the crystal, C-H⋯O inter-actions occur along the [100] direction.

4-(Naphthalene-2-carboxamido)-pyridin-1-ium thio-cyanate-N-(pyridin-4-yl)naphthalene-2-carboxamide (1/1).

The asymmetric unit of the title compound, C(16)H(13)N(2)O(+)·NCS(-)·C(16)H(12)N(2)O, contains two N-(pyridin-4-yl)naphthalene-2-carboxamide mol-ecules, both are partially protonated in the pyridine moiety, i.e. the H atom attached to the pyridine N atom is partially occupied with an occupancy factor of 0.61 (3) and 0.39 (3), respectively. In the crystal, protonated and neutral N-(pyridin-4-yl)naphthalene-2-carboxamide mol-ecules are linked by N-H⋯N hydrogen bonding; the thio-cyanate counter-ion links with both protonated and neutral N-(pyridin-4-yl)naphthalene-2-carboxamide mol-ecules via N-H⋯S and N-H⋯N hydrogen bonding. The dihedral angles between the pyridine ring and naphthalene ring systems are 11.33 (6) and 9.51 (6)°, respectively. π-π stacking is observed in the crystal structure, the shortest centroid-centroid distance being 3.5929 (8) Å. The crystal structure was determined from a nonmerohedral twin {ratio of the twin components = 0.357 (1):0.643 (1) and twin law [-100 0-10 -101]}.

Utilization of sacubitril/valsartan in patients with heart failure with reduced ejection fraction: real-world data from the ARIADNE registry.

AIMS: To compare baseline characteristics of patients with heart failure with reduced ejection fraction (HFrEF) initiated on sacubitril/valsartan compared with patients continued on conventional heart failure (HF)-treatment in a European out-patient setting. METHODS AND RESULTS: Between July 2016 and July 2019, ARIADNE enrolled 8787 outpatients aged ≥18 years with HFrEF from 17 European countries. Choice of therapy was solely at the investigators' discretion. In total, 4173 patients were on conventional HF-treatment (non-S/V group), while 4614 patients were on sacubitril/valsartan either at enrolment or started sacubitril/valsartan within 1 month of enrolment (S/V group). Of these, 2108 patients started sacubitril/valsartan treatment ±1 month around enrolment [restricted S/V (rS/V) group]. The average age of the patients was 68 years. Patients on S/V were more likely to have New York Heart Association (NYHA) class III or IV symptoms (50.3%, 44.6%, 32.1% in rS/V, S/V, and non-S/V, respectively) and had lower left ventricular ejection fraction (LVEF; 32.3%, 32.7%, and 35.4% in rS/V, S/V, and non-S/V, respectively; P < 0.0001). The most frequently received HF treatments were angiotensin-converting enzyme inhibitor/angiotensin receptor blocker (ACEI/ARB; ∼84% in non-S/V), followed by ß-blockers (∼80%) and mineralocorticoid receptor antagonists (MRAs; 53%). The use of triple HF therapy (ACEI/ARB/angiotensin receptor neprilysin inhibitor with ß-blockers and MRA) was higher in the S/V groups than non-S/V group (48.2%, 48.2%, and 40.2% in rS/V, S/V, and non-S/V, respectively). CONCLUSION: In this large multinational HFrEF registry, patients receiving sacubitril/valsartan tended to be younger with lower LVEF and higher NYHA class. Fewer than half of the patients received triple HF therapy.

Outcomes with sacubitril/valsartan in outpatients with heart failure and reduced ejection fraction: The ARIADNE registry.

AIMS: ARIADNE aimed to assess the association between effects of sacubitril/valsartan and no sacubitril/valsartan treatment and clinical characteristics, functional capacity, and clinical outcomes (cause-specific mortality and hospitalizations) in outpatients with heart failure (HF) with reduced ejection fraction (HFrEF). METHODS: ARIADNE was a prospective European registry of 9069 patients with HFrEF treated by office-based cardiologists or selected primary care physicians. Of the 8787 eligible for analysis, 4173 patients were on conventional HF treatment (non-S/V group), whereas 4614 patients were either on sacubitril/valsartan treatment at enrolment or started sacubitril/valsartan within 1 month of enrolment (S/V group). We also generated a restricted analysis set (rS/V) including only those 2108 patients who started sacubitril/valsartan treatment within the month prior to or after enrolment. RESULTS: At the baseline, average age of patients enrolled in the study was 68 years, and 23.9% (2099/8787) were female. At the baseline, the proportions of patients with New York Heart Association (NYHA) Class III symptoms were 30.9 (1288/4173), 42.8 (1974/4614), and 48.2% (1015/2108), in non-S/V, S/V, and rS/V groups, respectively. After 12 months of treatment, the proportion of patients with NYHA Class III at baseline who improved to Class II was 32.0% (290/907) in the non-S/V group vs. 46.3% (648/1399) in S/V group and 48.7% (349/717) in rS/V group. The overall mortality rate was 5.0 per 100 patient-years. Rates of HF hospitalizations were high (20.9, 20.3, and 21.2 per 100 patient-years in the non-S/V, S/V, and rS/V groups, respectively). Emergency room visits without hospitalization occurred in 3.9, 3.2, and 3.9% of patients in the non-S/V, S/V, and rS/V groups, respectively. CONCLUSIONS: This large HFrEF European registry provides a contemporary outcome profile of outpatients with HFrEF treated with or without sacubitril/valsartan. In a real-world setting, sacubitril/valsartan was associated with an improvement of symptoms in patients with HFrEF compared with the conventional HFrEF treatment.

1-(4,6-Dimethyl-pyrimidin-2-yl)-3-(3,5-dinitro-benzo-yl)thio-urea monohydrate.

The organic molecule in the title mol-ecule, C(14)H(12)N(6)O(5)S·H(2)O, is roughly planar with a maximum deviation of 0.156 (2) Å. An intra-molecular N-H⋯N hydrogen bond occurs. In the crystal, inter-molecular N-H⋯O and O-H⋯O hydrogen-bonding inter-actions connect the mol-ecules into a two-dimensional network that lies parallel to (101).