Annexin A1 attenuates microvascular complications through restoration of Akt signalling in a murine model of type 1 diabetes.

AIMS/HYPOTHESIS: Microvascular complications in the heart and kidney are strongly associated with an overall rise in inflammation. Annexin A1 (ANXA1) is an endogenous anti-inflammatory molecule that limits and resolves inflammation. In this study, we have used a bedside to bench approach to investigate: (1) ANXA1 levels in individuals with type 1 diabetes; (2) the role of endogenous ANXA1 in nephropathy and cardiomyopathy in experimental type 1 diabetes; and (3) whether treatment with human recombinant ANXA1 attenuates nephropathy and cardiomyopathy in a murine model of type 1 diabetes. METHODS: ANXA1 was measured in plasma from individuals with type 1 diabetes with or without nephropathy and healthy donors. Experimental type 1 diabetes was induced in mice by injection of streptozotocin (STZ; 45 mg/kg i.v. per day for 5 consecutive days) in C57BL/6 or Anxa1 -/- mice. Diabetic mice were treated with human recombinant (hr)ANXA1 (1 µg, 100 µl, 50 mmol/l HEPES; 140 mmol/l NaCl; pH 7.4, i.p.) or vehicle (100 µl, 50 mmol/l HEPES; 140 mmol/l NaCl; pH 7.4, i.p.). RESULTS: Plasma levels of ANXA1 were elevated in individuals with type 1 diabetes with/without nephropathy compared with healthy individuals (66.0 ± 4.2/64.0 ± 4 ng/ml vs 35.9 ± 2.3 ng/ml; p < 0.05). Compared with diabetic wild-type (WT) mice, diabetic Anxa1 -/- mice exhibited a worse diabetic phenotype and developed more severe cardiac (ejection fraction; 76.1 ± 1.6% vs 49.9 ± 0.9%) and renal dysfunction (proteinuria; 89.3 ± 5.0 µg/mg vs 113.3 ± 5.5 µg/mg). Mechanistically, compared with non-diabetic WT mice, the degree of the phosphorylation of mitogen-activated protein kinases (MAPKs) p38, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) was significantly higher in non-diabetic Anxa1 -/- mice in both the heart and kidney, and was further enhanced after STZ-induced type 1 diabetes. Prophylactic treatment with hrANXA1 (weeks 1-13) attenuated both cardiac (ejection fraction; 54.0 ± 1.6% vs 72.4 ± 1.0%) and renal (proteinuria; 89.3 ± 5.0 µg/mg vs 53.1 ± 3.4 µg/mg) dysfunction associated with STZ-induced diabetes, while therapeutic administration of hrANXA1 (weeks 8-13), after significant cardiac and renal dysfunction had already developed, halted the further functional decline in cardiac and renal function seen in diabetic mice administered vehicle. In addition, administration of hrANXA1 attenuated the increase in phosphorylation of p38, JNK and ERK, and restored phosphorylation of Akt in diabetic mice. CONCLUSIONS/INTERPRETATION: Overall, these results demonstrate that ANXA1 plasma levels are elevated in individuals with type 1 diabetes independent of a significant impairment in renal function. Furthermore, in mouse models with STZ-induced type 1 diabetes, ANXA1 protects against cardiac and renal dysfunction by returning MAPK signalling to baseline and activating pro-survival pathways (Akt). We propose ANXA1 to be a potential therapeutic option for the control of comorbidities in type 1 diabetes.

Cadherins: cellular adhesive molecules serving as signalling mediators.

The single pass, transmembrane proteins of the cadherin family have been appreciated as important proteins that regulate intercellular adhesion. In addition to this critical function, cadherins contribute to important signalling events that control cellular homeostasis. Many examples exist of classical, desmosomal and atypical cadherins participating in the regulation of signalling events that control homeostatic functions in cells. Much of the work on cadherin mediated signalling focuses on classical cadherins or on specific disease states such as pemphigus vulgaris. Cadherin mediated signalling has been shown to play critical roles during development, in proliferation, apoptosis, disease pathobiology and beyond. It is becoming increasingly clear that cadherins operate through a range of molecular mechanisms. The diversity of pathways and cellular functions regulated by cadherins suggests that we have only scratched the surface in terms of the roles that these versatile proteins play in signalling and cellular function.

Definition of a Novel Pathway Centered on Lysophosphatidic Acid To Recruit Monocytes during the Resolution Phase of Tissue Inflammation.

Blood-derived monocytes remove apoptotic cells and terminate inflammation in settings as diverse as atherosclerosis and Alzheimer's disease. They express high levels of the proresolving receptor ALX/FPR2, which is activated by the protein annexin A1 (ANXA1), found in high abundance in inflammatory exudates. Using primary human blood monocytes from healthy donors, we identified ANXA1 as a potent CD14(+)CD16(-) monocyte chemoattractant, acting via ALX/FPR2. Downstream signaling pathway analysis revealed the p38 MAPK-mediated activation of a calcium independent phospholipase A2 with resultant synthesis of lysophosphatidic acid (LPA) driving chemotaxis through LPA receptor 2 and actin cytoskeletal mobilization. In vivo experiments confirmed ANXA1 as an independent phospholipase A2-dependent monocyte recruiter; congruently, monocyte recruitment was significantly impaired during ongoing zymosan-induced inflammation in AnxA1(-/-) or alx/fpr2/3(-/-) mice. Using a dorsal air-pouch model, passive transfer of apoptotic neutrophils between AnxA1(-/-) and wild-type mice identified effete neutrophils as the primary source of soluble ANXA1 in inflammatory resolution. Together, these data elucidate a novel proresolving network centered on ANXA1 and LPA generation and identify previously unappreciated determinants of ANXA1 and ALX/FPR2 signaling in monocytes.

Regulation of intestinal epithelial intercellular adhesion and barrier function by desmosomal cadherin desmocollin-2.

The role of desmosomal cadherin desmocollin-2 (Dsc2) in regulating barrier function in intestinal epithelial cells (IECs) is not well understood. Here, we report the consequences of silencing Dsc2 on IEC barrier function in vivo using mice with inducible intestinal-epithelial-specific Dsc2 knockdown (KD) (Dsc2ERΔIEC). While the small intestinal gross architecture was maintained, loss of epithelial Dsc2 influenced desmosomal plaque structure, which was smaller in size and had increased intermembrane space between adjacent epithelial cells. Functional analysis revealed that loss of Dsc2 increased intestinal permeability in vivo, supporting a role for Dsc2 in the regulation of intestinal epithelial barrier function. These results were corroborated in model human IECs in which Dsc2 KD resulted in decreased cell-cell adhesion and impaired barrier function. It is noteworthy that Dsc2 KD cells exhibited delayed recruitment of desmoglein-2 (Dsg2) to the plasma membrane after calcium switch-induced intercellular junction reassembly, while E-cadherin accumulation was unaffected. Mechanistically, loss of Dsc2 increased desmoplakin (DP I/II) protein expression and promoted intermediate filament interaction with DP I/II and was associated with enhanced tension on desmosomes as measured by a Dsg2-tension sensor. In conclusion, we provide new insights on Dsc2 regulation of mechanical tension, adhesion, and barrier function in IECs.

Desmocollin-2 promotes intestinal mucosal repair by controlling integrin-dependent cell adhesion and migration.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a tight barrier, separating luminal antigens and microbes from underlying tissue compartments. Mucosal damage results in a compromised epithelial barrier that can lead to excessive immune responses as observed in inflammatory bowel disease. Efficient wound repair is critical to reestablish the mucosal barrier and homeostasis. Intestinal epithelial cells (IEC) exclusively express the desmosomal cadherins, Desmoglein-2 and Desmocollin-2 (Dsc2) that contribute to mucosal homeostasis by strengthening intercellular adhesion between cells. Despite this important property, specific contributions of desmosomal cadherins to intestinal mucosal repair after injury remain poorly investigated in vivo. Here we show that mice with inducible conditional knockdown (KD) of Dsc2 in IEC (Villin-CreERT2; Dsc2 fl/fl) exhibited impaired mucosal repair after biopsy-induced colonic wounding and recovery from dextran sulfate sodium-induced colitis. In vitro analyses using human intestinal cell lines after KD of Dsc2 revealed delayed epithelial cell migration and repair after scratch-wound healing assay that was associated with reduced cell-matrix traction forces, decreased levels of integrin ß1 and ß4, and altered activity of the small GTPase Rap1. Taken together, these results demonstrate that epithelial Dsc2 is a key contributor to intestinal mucosal wound healing in vivo.

Ucma/GRP inhibits phosphate-induced vascular smooth muscle cell calcification via SMAD-dependent BMP signalling.

Vascular calcification (VC) is the process of deposition of calcium phosphate crystals in the blood vessel wall, with a central role for vascular smooth muscle cells (VSMCs). VC is highly prevalent in chronic kidney disease (CKD) patients and thought, in part, to be induced by phosphate imbalance. The molecular mechanisms that regulate VC are not fully known. Here we propose a novel role for the mineralisation regulator Ucma/GRP (Upper zone of growth plate and Cartilage Matrix Associated protein/Gla Rich Protein) in phosphate-induced VSMC calcification. We show that Ucma/GRP is present in calcified atherosclerotic plaques and highly expressed in calcifying VSMCs in vitro. VSMCs from Ucma/GRP-/- mice showed increased mineralisation and expression of osteo/chondrogenic markers (BMP-2, Runx2, ß-catenin, p-SMAD1/5/8, ALP, OCN), and decreased expression of mineralisation inhibitor MGP, suggesting that Ucma/GRP is an inhibitor of mineralisation. Using BMP signalling inhibitor noggin and SMAD1/5/8 signalling inhibitor dorsomorphin we showed that Ucma/GRP is involved in inhibiting the BMP-2-SMAD1/5/8 osteo/chondrogenic signalling pathway in VSMCs treated with elevated phosphate concentrations. Additionally, we showed for the first time evidence of a direct interaction between Ucma/GRP and BMP-2. These results demonstrate an important role of Ucma/GRP in regulating osteo/chondrogenic differentiation and phosphate-induced mineralisation of VSMCs.

Pharmacological Treatment with Annexin A1 Reduces Atherosclerotic Plaque Burden in LDLR-/- Mice on Western Type Diet.

OBJECTIVE: To investigate therapeutic effects of annexin A1 (anxA1) on atherogenesis in LDLR-/- mice. METHODS: Human recombinant annexin A1 (hr-anxA1) was produced by a prokaryotic expression system, purified and analysed on phosphatidylserine (PS) binding and formyl peptide receptor (FPR) activation. Biodistribution of 99mTechnetium-hr-anxA1 was determined in C57Bl/6J mice. 12 Weeks old LDLR-/- mice were fed a Western Type Diet (WTD) during 6 weeks (Group I) or 12 weeks (Group P). Mice received hr-anxA1 (1 mg/kg) or vehicle by intraperitoneal injection 3 times per week for a period of 6 weeks starting at start of WTD (Group I) or 6 weeks after start of WTD (Group P). Total aortic plaque burden and phenotype were analyzed using immunohistochemistry. RESULTS: Hr-anxA1 bound PS in Ca2+-dependent manner and activated FPR2/ALX. It inhibited rolling and adherence of neutrophils but not monocytes on activated endothelial cells. Half lives of circulating 99mTc-hr-anxA1 were <10 minutes and approximately 6 hours for intravenously (IV) and intraperitoneally (IP) administered hr-anxA1, respectively. Pharmacological treatment with hr-anxA1 had no significant effect on initiation of plaque formation (-33%; P = 0.21)(Group I) but significantly attenuated progression of existing plaques of aortic arch and subclavian artery (plaque size -50%, P = 0.005; necrotic core size -76% P = 0.015, hr-anxA1 vs vehicle) (Group P). CONCLUSION: Hr-anxA1 may offer pharmacological means to treat chronic atherogenesis by reducing FPR-2 dependent neutrophil rolling and adhesion to activated endothelial cells and by reducing total plaque inflammation.

Molecular imaging to identify the vulnerable plaque--from basic research to clinical practice.

Cardiovascular disease (CVD) is still the leading cause of death in the Western World. Adverse outcomes of CVD include stroke, myocardial infarction, and heart failure. Atherosclerosis is considered to be the major cause of CVD and is estimated to cause half of all deaths in developed countries. Atherosclerotic lesions of the vessel wall may obstruct blood flow mechanically through stenosis, but rupture of atherosclerotic plaques causing formation of occlusive thrombi is far more prevalent. Unfortunately, conventional diagnostic tools fail to assess whether a plaque is vulnerable to rupture. Research over the past decade identified the biological processes that are implicated in the course towards plaque rupture, like cell death and inflammation. Knowledge about plaque biology propelled the development of imaging techniques that target biologic processes in order to predict the vulnerable plaque. This paper discusses novel and existing molecular imaging targets and addresses advantages and disadvantages of these targets and respective imaging techniques in respect of clinical application and socio-economic impact.