Assessing Efficacy of Afatinib toward Elastic Matrix Repair in Aortic Aneurysms.

Abdominal aortic aneurysm (AAA) is a critical, multifactorial cardiovascular disorder marked by localized dilatation of the abdominal aorta. A major challenge to countering the pathophysiology of AAAs lies in the naturally irreversible breakdown of elastic fibers in the aorta wall, which is linked to the poor elastogenicity of adult and diseased vascular smooth muscle cells (SMCs) and their impaired ability to assemble mature elastic fibers in a chronic proteolytic tissue milieu. We have previously shown that these are downstream effects of neutrophil elastase-induced activation of the epidermal growth factor receptor (EGFR) activity in aneurysmal SMCs. The novelty of this study lies in investigating the benefits of an EGFR inhibitor drug, afatinib (used to treat nonsmall cell lung cancer), for proelastogenic and antiproteolytic stimulation of aneurysmal SMCs. In in vitro cell cultures, we have shown that safe doses of 0.5 and 1 nM afatinib inhibit EGFR and p-extracellular signal-regulated kinases 1/2 protein expression by 50-70% and downstream elastolytic matrix metalloprotease 2 (MMP2) versus untreated control cultures. In addition, elastin production on a per cell basis was significantly upregulated by afatinib doses within the 0.1-1 nM dose range, which was further validated through transmission electron microscopy showing significantly increased presence of tropoelastin coacervates and maturing elastic fibers upon afatinib treatment at the above doses. Therefore, our studies for the first time demonstrate the therapeutic benefits of afatinib toward use for elastic matrix repair in small AAAs.

Targeting Epidermal Growth Factor Receptor to Stimulate Elastic Matrix Regenerative Repair.

Abdominal aortic aneurysms (AAAs) represent a multifactorial, proteolytic disorder involving disintegration of the matrix structure within the AAA wall. Intrinsic deficiency of adult vascular cells to regenerate and repair the wall elastic matrix, which contributes to vessel stretch and recoil, is a major clinical challenge to therapeutic reversal of AAA growth. In this study, we investigate the involvement of epidermal growth factor receptor-mitogen activated protein kinase (EGFR-MAPK) pathway in the activation of aneurysmal smooth muscle cells (SMCs) by neutrophil elastase, and how EGFR can be targeted for elastic matrix regeneration. We have demonstrated that neutrophil elastase activates EGFR and downregulates expression level of key elastin homeostasis genes (elastin, crosslinking enzyme-lysyl oxidase, and fibulin4) between a dose range of 1-10 µg/mL (p < 0.05). It also incites downstream proteolytic outcomes by upregulating p-extracellular signal-regulated kinase (ERK)1/2 (p < 0.0001) and matrix metalloprotease 2 (MMP2) at a protein level, which is significantly downregulated upon EGFR-specific inhibition by tyrosine kinase inhibitor AG1478 (p-ERK1/2 and MMP2 [p < 0.05]). Moreover, we have shown that EGFR inhibition suppresses collagen amounts in aneurysmal SMCs (p < 0.05) and promotes robust formation of elastic fibers by enhancing its deposition in the extracellular space. Hence, the EGFR-MAPK pathway in aneurysmal cells can be targeted to provide therapeutic effects toward stimulating vascular matrix regeneration. Impact statement Proteolytic disorders such as aortal expansions, called abdominal aortic aneurysms (AAAs), are characterized by naturally irreversible enzymatic breakdown and loss of elastic fibers, a problem that has not yet been surmounted by existing tissue engineering approaches. In this work, we show, for the first time, how epidermal growth factor receptor (EGFR) inhibition provides downstream benefits in elastic fiber assembly and deposition in aneurysmal smooth muscle cell cultures. This work can open future possibilities for development of EGFR-targeted drug-based therapies not only for vessel wall repair in AAAs but also other proteolytically compromised elastic tissues.

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.

Adult Mesenchymal Stem Cells and Derivatives in Improved Elastin Homeostasis in a Rat Model of Abdominal Aortic Aneurysms.

Abdominal aortic aneurysms (AAAs) are localized rupture-prone expansions of the aorta with limited reversibility that develop due to proteolysis of the elastic matrix. Natural regenerative repair of an elastic matrix is difficult due to the intrinsically poor elastogenicity of adult vascular smooth muscle cells (VSMCs). This justifies the need to provide external, pro-elastin regenerative- and anti-proteolytic stimuli to VSMCs in the AAA wall towards reinstating matrix structure in the aorta wall. Introducing alternative phenotypes of highly elastogenic and contractile cells into the AAA wall capable of providing such cues, proffers attractive prospects for AAA treatment. In this regard, we have previously demonstrated the superior elastogenicity of bone marrow mesenchymal stem cell (BM-MSC)-derived SMCs (cBM-SMCs) and their ability to provide pro-elastogenic and anti-proteolytic stimuli to aneurysmal SMCs in vitro. However, the major issues associated with cell therapy, such as their natural ability to home into the AAA tissue, their in vivo biodistribution and retention in the AAA wall, and possible paracrine effects on AAA tissue repair processes in the event of localization in remote tissues remain uncertain. Therefore, in this study we focused on assessing the fate, safety, and AAA reparative effects of BM-MSC-derived cBM-SMCs in vivo. Our results indicate that the cBM-SMCs (a) possess natural homing abilities similar to the undifferentiated BM-MSCs, (b) exhibit higher retention upon localization in the aneurysmal aorta than BM-MSCs, (c) downregulate the expression of several inflammatory and pro-apoptotic cytokines that are upregulated in the AAA wall contributing to accelerated elastic matrix breakdown and suppression of elastic fiber neo-assembly, repair, and crosslinking, and (d) improve elastic matrix content and structure in the AAA wall toward slowing the growth of AAAs. Our study provides initial evidence of the in vivo elastic matrix reparative benefits of cBM-SMCs and their utility in cell therapy to reverse the pathophysiology of AAAs.