JNK2 silencing lipid nanoparticles for elastic matrix repair.

The over-expression of c-Jun N-terminal kinase (JNK2), a stress activated mitogen kinase, in the aortic wall plays a critical role in the formation and progression of abdominal aortic aneurysm (AAA). This triggers chronic downstream upregulation of elastolytic matrix metalloproteinases (MMPs), MMPs2 and 9 to cause progressive proteolytic breakdown of the wall elastic matrix. We have previously shown that siNRA knockdown of JNK2 gene expression in an AAA culture model stimulates downstream elastin gene expression, elastic fiber formation, crosslinking and reduces elastolytic MMPs2 and 9. Since naked siRNA poorly routes to intracellular targets, has poor stability in blood, and could be potentially toxic and immunogenic, this project is aimed to develop PEGylated lipid nanoparticles (LNPs) for delivery of JNK siRNA and to generate evidence of successful JNK2 knockdown and downstream attenuation of MMP2 gene and protein expressions. LNPs were formulated using thin-film hydration technique and had the size of 100-200 nm with zeta-potential ranging between 30 and 40 mV. JNK siRNA loaded PEGylated LNPs successfully knocked down JNK2 in cytokine-activated rat aneurysmal smooth muscle (EaRASMC) cultures. This resulted in a downstream decrease in MMP2 gene and protein expression and an upward trend in expression of genes for proteins critical for elastic fiber assembly such as elastin (ELN) and lysyl oxidase (LOX). Our result indicates cationic LNPs to be potential carriers for JNK siRNA delivery improving potency for elastin homeostasis required for AAA repair which could possibly provide benefits in preventing the progression of small AAAs.

Sodium Nitroprusside Stimulation of Elastic Matrix Regeneration by Aneurysmal Smooth Muscle Cells.

The chronic overexpression of matrix metalloproteases leading to consequent degradation and loss of the elastic matrix with the reduction in tissue elasticity is central to the pathophysiology of proteolytic disorders, such as abdominal aortic aneurysms (AAAs), which are localized rupture-prone aortic expansions. Effecting tissue repair to alleviate this condition is contingent on restoring elastic matrix homeostasis in the aortic wall. This is naturally irreversible due to the poor elastogenicity of adult and diseased vascular cells, and the impaired ability to assemble mature elastic fibers, more so in the context of phenotypic changes to medial smooth muscle cells (SMCs) owing to the loss of nitric oxide (NO) signaling in the AAA wall tissue. In this study, we report the benefits of the exposure of primary human aneurysmal SMCs (aHASMCs) to NO donor drug, sodium nitroprusside (SNP), in improving extracellular matrix homeostasis, particularly aspects of elastic fiber assembly, and inhibition of proteolytic degradation. SNP treatment (100 nM) upregulated elastic matrix regeneration at both gene (p < 0.05) and protein levels (p < 0.01) without affecting cell proliferation, improved gene, and protein expression of crosslinking enzyme, lysyl oxidase (p < 0.05), inhibited the expression of MMP2 (matrix metalloprotease 2) significantly (p < 0.05) and promoted contractile SMC phenotypes in aHASMC culture. In addition, SNP also attenuated the expression of mitogen-activated protein kinases, a significant player in AAA formation and progression. Our results indicate the promise of SNP for therapeutic augmentation of elastic matrix regeneration, with prospects for wall repair in AAAs. Impact Statement Chronic and naturally irreversible enzymatic degradation and loss of elastic fibers are centric to proteolytic disorders such as abdominal aortic aneurysms (AAAs). This is linked to poor elastogenicity of adult and diseased vascular cells, compromising their ability to assemble mature elastic fibers. Toward addressing this, we demonstrate the phenotype-modulatory properties of a nitric oxide donor drug, sodium nitroprusside on aneurysmal smooth muscle cells, and its dose-specific proelastogenic and antiproteolytic properties for restoring elastic matrix homeostasis. Combined with the development of vehicles for site-localized, controlled drug delivery, this can potentially lead to a new nonsurgical approach for AAA wall repair in the future.

Stem Cell Based Approaches to Modulate the Matrix Milieu in Vascular Disorders.

The extracellular matrix (ECM) represents a complex and dynamic framework for cells, characterized by tissue-specific biophysical, mechanical, and biochemical properties. ECM components in vascular tissues provide structural support to vascular cells and modulate their function through interaction with specific cell-surface receptors. ECM-cell interactions, together with neurotransmitters, cytokines, hormones and mechanical forces imposed by blood flow, modulate the structural organization of the vascular wall. Changes in the ECM microenvironment, as in post-injury degradation or remodeling, lead to both altered tissue function and exacerbation of vascular pathologies. Regeneration and repair of the ECM are thus critical toward reinstating vascular homeostasis. The self-renewal and transdifferentiating potential of stem cells (SCs) into other cell lineages represents a potentially useful approach in regenerative medicine, and SC-based approaches hold great promise in the development of novel therapeutics toward ECM repair. Certain adult SCs, including mesenchymal stem cells (MSCs), possess a broader plasticity and differentiation potential, and thus represent a viable option for SC-based therapeutics. However, there are significant challenges to SC therapies including, but not limited to cell processing and scaleup, quality control, phenotypic integrity in a disease milieu in vivo, and inefficient delivery to the site of tissue injury. SC-derived or -inspired strategies as a putative surrogate for conventional cell therapy are thus gaining momentum. In this article, we review current knowledge on the patho-mechanistic roles of ECM components in common vascular disorders and the prospects of developing adult SC based/inspired therapies to modulate the vascular tissue environment and reinstate vessel homeostasis in these disorders.