Angiotensin Receptor-Neprilysin Inhibition (Sacubitril/Valsartan) Reduces Structural Arterial Stiffness in Middle-Aged Mice.

BACKGROUND: Increasing arterial stiffness is a prominent feature of the aging cardiovascular system. Arterial stiffening leads to fundamental alterations in central hemodynamics with widespread detrimental implications for organ function resulting in significant morbidity and death, and specific therapies to address the underlying age-related structural arterial remodeling remain elusive. The present study investigates the potential of the recently clinically available dual angiotensin receptor-neprilysin inhibitor (ARNI) sacubitril/valsartan (LCZ696) to counteract age-related arterial fibrotic remodeling and stiffening in 1-year-old mice. METHODS AND RESULTS: Treatment of in 1-year-old mice with ARNI (sacubitril/valsartan), in contrast to angiotensin receptor blocker monotherapy (valsartan) and vehicle treatment (controls), significantly decreases structural aortic stiffness (as measured by in vivo pulse-wave velocity and ex vivo aortic pressure myography). This phenomenon appears, at least partly, independent of (indirect) blood pressure effects and may be related to a direct antifibrotic interference with aortic smooth muscle cell collagen production. Furthermore, we find aortic remodeling and destiffening due to ARNI treatment to be associated with improved parameters of cardiac diastolic function in aged mice. CONCLUSIONS: This study provides preclinical mechanistic evidence indicating that ARNI-based interventions may counteract age-related arterial stiffening and may therefore be further investigated as a promising strategy to improve cardiovascular outcomes in the elderly.

MicroRNA miR-29b regulates diabetic aortic remodeling and stiffening.

Patients with type 2 diabetes (T2D) are threatened by excessive cardiovascular morbidity and mortality. While accelerated arterial stiffening may represent a critical mechanistic factor driving cardiovascular risk in T2D, specific therapies to contain the underlying diabetic arterial remodeling have been elusive. The present translational study investigates the role of microRNA-29b (miR-29b) as a driver and therapeutic target of diabetic aortic remodeling and stiffening. Using a murine model (db/db mice), as well as human aortic tissue samples, we find that diabetic aortic remodeling and stiffening is associated with medial fibrosis, as well as fragmentation of aortic elastic layers. miR-29b is significantly downregulated in T2D and miR-29b repression is sufficient to induce both aortic medial fibrosis and elastin breakdown through upregulation of its direct target genes COL1A1 and MMP2 thereby increasing aortic stiffness. Moreover, antioxidant treatment restores aortic miR-29b levels and counteracts diabetic aortic remodeling. Concluding, we identify miR-29b as a comprehensive-and therefore powerful-regulator of aortic remodeling and stiffening in T2D that moreover qualifies as a (redox-sensitive) target for therapeutic intervention.

IL-1ß-Induced Downregulation of the Multifunctional PDZ Adaptor PDZK1 Is Attenuated by ERK Inhibition, RXRα, or PPARα Stimulation in Enterocytes.

Background: The PDZ adaptor protein PDZK1 modulates the membrane expression and function of a variety of intestinal receptors and ion/nutrient transporters. Its expression is strongly decreased in inflamed intestinal mucosa of mice and IBD patients. Aim and Methods: We investigated whether the inflammation-associated PDZK1 downregulation is a direct consequence of proinflammatory cytokine release by treating intestinal Caco-2BBE cells with TNF-α, IFN-γ, and IL-1ß, and analysing PDZK1 promotor activity, mRNA and protein expression. Results: IL-1ß was found to significantly decrease PDZK1 promoter activity, mRNA and protein expression in Caco-2BBE cells. A distal region of the hPDZK1 promoter was identified to be important for basal expression and IL-1ß-responsiveness. This region harbors the retinoid acid response element RARE as well as binding sites for transcription factors involved in IL-ß downstream signaling. ERK1/2 inhibition by the specific MEK1/2 inhibitors PD98059/U0126 significantly attenuated the IL-1ß mediated downregulation of PDZK1, while NF-κB, p38 MAPK, and JNK inhibition did not. Expression of the nuclear receptors RXRα and PPARα was decreased in inflamed colonic-mucosa of ulcerative colitis patients and in IL-1ß-treated Caco2-BBE cells. Moreover, the RAR/RXR ligand 9-cis retinoic acid and the PPARα-agonist GW7647 stimulated PDZK1 mRNA and protein expression and attenuated IL-1ß-mediated inhibition. Conclusions: The strong decrease in PDZK1 expression during intestinal inflammation may be in part a consequence of IL-1ß-mediated RXRα and PPARα repression and can be attenuated by agonists for either nuclear receptor, or by ERK1/2 inhibition. The negative consequences of inflammation-induced PDZK1 downregulation on epithelial transport-function may thus be amenable to pharmacological therapy.

Na+ /H+ exchanger NHE1 and NHE2 have opposite effects on migration velocity in rat gastric surface cells.

Following superficial injury, neighbouring gastric epithelial cells close the wound by rapid cell migration, a process called epithelial restitution. Na+ /H+ exchange (NHE) inhibitors interfere with restitution, but the role of the different NHE isoforms expressed in gastric pit cells has remained elusive. The role of the basolaterally expressed NHE1 (Slc9a1) and the presumably apically expressed NHE2 (Slc9a2) in epithelial restitution was investigated in the nontransformed rat gastric surface cell line RGM1. Migration velocity was assessed by loading the cells with the fluorescent dye DiR and following closure of an experimental wound over time. Since RGM1 cells expressed very low NHE2 mRNA and have low transport activity, NHE2 was introduced by lentiviral gene transfer. In medium with pH 7.4, RGM1 cells displayed slow wound healing even in the absence of growth factors and independently of NHE activity. Growth factors accelerated wound healing in a partly NHE1-dependent fashion. Preincubation with acidic pH 7.1 stimulated restitution in a NHE1-dependent fashion. When pH 7.1 was maintained during the restitution period, migratory speed was reduced to ∼10% of the speed at pH 7,4, and the residual restitution was further inhibited by NHE1 inhibition. Lentiviral NHE2 expression increased the steady-state pHi and reduced the restitution velocity after low pH preincubation, which was reversible by pharmacological NHE2 inhibition. The results demonstrate that in RGM1 cells, migratory velocity is increased by NHE1 activation, while NHE2 activity inhibit this process. A differential activation of NHE1 and NHE2 may therefore, play a role in the initiation and completion of the epithelial restitution process.

Evidence for a causal link between adaptor protein PDZK1 downregulation and Na⁺/H⁺ exchanger NHE3 dysfunction in human and murine colitis.

A dysfunction of the Na(+)/H(+) exchanger isoform 3 (NHE3) significantly contributes to the reduced salt absorptive capacity of the inflamed intestine. We previously reported a strong decrease in the NHERF family member PDZK1 (NHERF3), which binds to NHE3 and regulates its function in a mouse model of colitis. The present study investigates whether a causal relationship exists between the decreased PDZK1 expression and the NHE3 dysfunction in human and murine intestinal inflammation. Biopsies from the colon of patients with ulcerative colitis, murine inflamed ileal and colonic mucosa, NHE3-transfected Caco-2BBe colonic cells with short hairpin RNA (shRNA) knockdown of PDZK1, and Pdzk1-gene-deleted mice were studied. PDZK1 mRNA and protein expression was strongly decreased in inflamed human and murine intestinal tissue as compared to inactive disease or control tissue, whereas that of NHE3 or NHERF1 was not. Inflamed human and murine intestinal tissues displayed correct brush border localization of NHE3 but reduced acid-activated NHE3 transport activity. A similar NHE3 transport defect was observed when PDZK1 protein content was decreased by shRNA knockdown in Caco-2BBe cells or when enterocyte PDZK1 protein content was decreased to similar levels as found in inflamed mucosa by heterozygote breeding of Pdzk1-gene-deleted and WT mice. We conclude that a decrease in PDZK1 expression, whether induced by inflammation, shRNA-mediated knockdown, or heterozygous breeding, is associated with a decreased NHE3 transport rate in human and murine enterocytes. We therefore hypothesize that inflammation-induced loss of PDZK1 expression may contribute to the NHE3 dysfunction observed in the inflamed intestine.