PACAP regulates VPAC1 expression, inflammatory processes and lipid homeostasis in M1- and M2-macrophages.

Background: Pituitary adenylate cyclase-activating polypeptide (PACAP) acts as an anti-atherogenic neuropeptide and plays an important role in cytoprotective, as well as inflammatory processes, and cardiovascular regulation. Therefore, the aim of this study is to investigate the regulatory effects of PACAP and its receptor VPAC1 in relation to inflammatory processes and lipid homeostasis in different macrophage (MΦ) subtypes. Methods: To investigate the role of PACAP deficiency in the pathogenesis of atherosclerosis under standard chow (SC) or cholesterol-enriched diet (CED) in vivo, PACAP-/- mice were crossbred with ApoE-/- to generate PACAP-/-/ApoE-/- mice. Lumen stenosis in the aortic arch and different MΦ-subtypes were analyzed in atherosclerotic plaques by quantitative immunohistochemistry. Undifferentiated bone marrow-derived cells (BMDC) from 30-weeks-old ApoE-/- and PACAP-/-/ApoE-/- mice were isolated, differentiated into BMDM1- and BMDM2-MΦ, and incubated with oxidized low-density lipoprotein (oxLDL). In addition, PMA-differentiated human THP-1 MΦ were further differentiated into M1-/M2-MΦ and subsequently treated with PACAP38, the VPAC1 agonist [(Ala11,22,28)VIP], the antagonist (PG 97-269), and/or oxLDL. Uptake/accumulation of oxLDL was analyzed by oxLDL-DyLight™488 and Bodipy™ 493/503. The mRNA expression was analyzed by qRT-PCR, protein levels by Western blot, and cytokine release by ELISA. Results: In vivo, after 30 weeks of SC, PACAP-/-/ApoE-/- mice showed increased lumen stenosis compared with ApoE-/- mice. In atherosclerotic plaques of PACAP-/-/ApoE-/- mice under CED, immunoreactive areas of VPAC1, CD86, and CD163 were increased compared with ApoE-/- mice. In vitro, VPAC1 protein levels were increased in PACAP-/-/ApoE-/- BMDM compared with ApoE-/- BMDM, resulting in increased TNF-α mRNA expression in BMDM1-MΦ and decreased TNF-α release in BMDM2-MΦ. Concerning lipid homeostasis, PACAP deficiency decreased the area of lipid droplets in BMDM1-/M2-MΦ with concomitant increasing adipose differentiation-related protein level. In THP-1 M1-/M2-MΦ, the VPAC1 antagonist increased the uptake of oxLDL, whereas the VPAC1 agonist decreased the oxLDL-induced intracellular triglyceride content. Conclusion: Our data suggest that PACAP via VPAC1 signaling plays an important regulatory role in inflammatory processes in atherosclerotic plaques and in lipid homeostasis in different MΦ-subtypes, thereby affecting foam cell formation. Therefore, VPAC1 agonists or PACAP may represent a new class of anti-atherogenic therapeutics.

Pre-existing anti-drug antibodies in Fabry disease show less affinity for pegunigalsidase alfa.

We analyzed the cross-reactivity of anti-drug antibodies (ADAs) against agalsidase-alfa and -beta from 49 patients with Fabry disease (FD) against the novel PEGylated enzyme pegunigalsidase-alfa (PRX-102). The affinity of purified anti-AGAL antibodies from pooled patient sera was significantly lower for PRX-102 compared to agalsidase-alfa and -beta (both p < 0.05). Pull-down experiments revealed the presence of masked epitopes on PRX-102, possibly due to PEGylation. ADA titers in serum (µg/mL) and corresponding inhibitory capacities against agalsidase-alfa and -beta were measured in male patients with FD, showing strong correlations (r2 = 0.9978 and 0.4930, both p < 0.001). Affinities of ADAs of individual patients against PRX-102 (Kd: 3.55 ± 2.72 µmol) were significantly lower compared to agalsidase alfa (Kd: 1.99 ± 1.26 µmol) and -beta (Kd: 2.18 ± 1.51 µmol) (both p < 0.0001). Cross-ELISAs supported the presence of masked epitopes on PRX-102. Importantly, inhibition measurements also revealed a 30% reduction in inhibitory capacity of pre-existing ADAs towards PRX-102. Enzyme-uptake experiments in AGAL-deficient EA.hy926 cells demonstrated less effects of ADAs on cellular PRX-102 uptake compared with agalsidase beta. We conclude that due to the reduced affinity of pre-existing ADAs against agalsidase-alfa or -beta, ADA-affected patients might benefit from a therapy switch to PRX-102, which is currently evaluated in clinical trials.

Endothelial Dysfunction in Fabry Disease Is Related to Glycocalyx Degradation.

Fabry disease (FD) is an X-linked multisystemic lysosomal storage disease due to a deficiency of α-galactosidase A (GLA/AGAL). Progressive cellular accumulation of the AGAL substrate globotriaosylceramide (Gb3) leads to endothelial dysfunction. Here, we analyzed endothelial function in vivo and in vitro in an AGAL-deficient genetic background to identify the processes underlying this small vessel disease. Arterial stiffness and endothelial function was prospectively measured in five males carrying GLA variants (control) and 22 FD patients under therapy. AGAL-deficient endothelial cells (EA.hy926) and monocytes (THP1) were used to analyze endothelial glycocalyx structure, function, and underlying inflammatory signals. Glycocalyx thickness and small vessel function improved significantly over time (p<0.05) in patients treated with enzyme replacement therapy (ERT, n=16) and chaperones (n=6). AGAL-deficient endothelial cells showed reduced glycocalyx and increased monocyte adhesion (p<0.05). In addition, increased expression of angiopoietin-2, heparanase and NF-κB was detected (all p<0.05). Incubation of wild-type endothelial cells with pathological globotriaosylsphingosine concentrations resulted in comparable findings. Treatment of AGAL-deficient cells with recombinant AGAL (p<0.01), heparin (p<0.01), anti-inflammatory (p<0.001) and antioxidant drugs (p<0.05), and a specific inhibitor (razuprotafib) of angiopoietin-1 receptor (Tie2) (p<0.05) improved glycocalyx structure and endothelial function in vitro. We conclude that chronic inflammation, including the release of heparanases, appears to be responsible for the degradation of the endothelial glycocalyx and may explain the endothelial dysfunction in FD. This process is partially reversible by FD-specific and anti-inflammatory treatment, such as targeted protective Tie2 treatment.

α-Galactosidase a Deficiency in Fabry Disease Leads to Extensive Dysregulated Cellular Signaling Pathways in Human Podocytes.

Fabry disease (FD) is caused by mutations in the α-galactosidase A (GLA) gene encoding the lysosomal AGAL enzyme. Loss of enzymatic AGAL activity and cellular accumulation of sphingolipids (mainly globotriaosylcermide) may lead to podocyturia and renal loss of function with increased cardiovascular morbidity and mortality in affected patients. To identify dysregulated cellular pathways in FD, we established a stable AGAL-deficient podocyte cell line to perform a comprehensive proteome analysis. Imbalanced protein expression and function were analyzed in additional FD cell lines including endothelial, epithelial kidney, patient-derived urinary cells and kidney biopsies. AGAL-deficient podocytes showed dysregulated proteins involved in thermogenesis, lysosomal trafficking and function, metabolic activity, cell-cell interactions and cell cycle. Proteins associated with neurological diseases were upregulated in AGAL-deficient podocytes. Rescues with inducible AGAL expression only partially normalized protein expression. A disturbed protein expression was confirmed in endothelial, epithelial and patient-specific cells, pointing toward fundamental pathway disturbances rather than to cell type-specific alterations in FD. We conclude that a loss of AGAL function results in profound changes of cellular pathways, which are ubiquitously in different cell types. Due to these profound alterations, current approved FD-specific therapies may not be sufficient to completely reverse all dysregulated pathways.