An Elastin-like Polypeptide-fusion peptide targeting capsid-tegument interface as an antiviral against cytomegalovirus infection.

The tegument protein pp150 of Human Cytomegalovirus (HCMV) is known to be essential for the final stages of virus maturation and mediates its functions by interacting with capsid proteins. Our laboratory has previously identified the critical regions in pp150 important for pp150-capsid interactions and designed peptides similar in sequence to these regions, with a goal to competitively inhibit capsid maturation. Treatment with a specific peptide (PepCR2 or P10) targeted to pp150 conserved region 2 led to a significant reduction in murine CMV (MCMV) growth in cell culture, paving the way for in vivo testing in a mouse model of CMV infection. However, the general pharmacokinetic parameters of peptides, including rapid degradation and limited tissue and cell membrane permeability, pose a challenge to their successful use in vivo. Therefore, we designed a biopolymer-stabilized elastin-like polypeptide (ELP) fusion construct (ELP-P10) to enhance the bioavailability of P10. Antiviral efficacy and cytotoxic effects of ELP-P10 were studied in cell culture, and pharmacokinetics, biodistribution, and antiviral efficacy were studied in a mouse model of CMV infection. ELP-P10 maintained significant antiviral activity in cell culture, and this conjugation significantly enhanced P10 bioavailability in mouse tissues. The fluorescently labeled ELP-P10 accumulated to higher levels in mouse liver and kidneys as compared to the unconjugated P10. Moreover, viral titers from vital organs of MCMV-infected mice indicated a significant reduction of virus load upon ELP-P10 treatment. Therefore, ELP-P10 has the potential to be developed into an effective antiviral against CMV infection.

Fibroblasts' secretome from calcified and non-calcified dermis in Pseudoxanthoma elasticum differently contributes to elastin calcification.

Pseudoxanthoma elasticum (PXE) is a rare disease characterized by ectopic calcification, however, despite the widely spread effect of pro/anti-calcifying systemic factors associated with this genetic metabolic condition, it is not known why elastic fibers in the same patient are mainly fragmented or highly mineralized in clinically unaffected (CUS) and affected (CAS) skin, respectively. Cellular morphology and secretome are investigated in vitro in CUS and CAS fibroblasts. Here we show that, compared to CUS, CAS fibroblasts exhibit: a) differently distributed and organized focal adhesions and stress fibers; b) modified cell-matrix interactions (i.e., collagen gel retraction); c) imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases; d) differentially expressed pro- and anti-calcifying proteoglycans and elastic-fibers associated glycoproteins. These data emphasize that in the development of pathologic mineral deposition fibroblasts play an active role altering the stability of elastic fibers and of the extracellular matrix milieu creating a local microenvironment guiding the level of matrix remodeling at an extent that may lead to degradation (in CUS) or to degradation and calcification (in CAS) of the elastic component. In conclusion, this study contributes to a better understanding of the mechanisms of the mineral deposition that can be also associated with several inherited or age-related diseases (e.g., diabetes, atherosclerosis, chronic kidney diseases).

Elastin-derived peptides favor type 2 innate lymphoid cells in chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a condition characterized by chronic airway inflammation and obstruction, primarily caused by tobacco smoking. Although the involvement of immune cells in COPD pathogenesis is well established, the contribution of innate lymphoid cells (ILCs) remains poorly understood. ILCs are a type of innate immune cells that participate in tissue remodeling processes, but their specific role in COPD has not been fully elucidated. During COPD, the breakdown of pulmonary elastin generates elastin peptides that elicit biological activities on immune cells. This study aimed to investigate the presence of ILC in patients with COPD and examine the impact of elastin peptides on their functionality. Our findings revealed an elevated proportion of ILC2 in the peripheral blood of patients with COPD, and a general activation of ILC as indicated by an increase in their cytokine secretion capacity. Notably, our study demonstrated that serum from patients with COPD promotes ILC2 phenotype, likely due to the elevated concentration of IL-5, a cytokine known to favor ILC2 activation. Furthermore, we uncovered that this increase in IL-5 secretion is partially attributed to its secretion by macrophages upon stimulation by elastin peptides, suggesting an indirect role of elastin peptides on ILC in COPD. These findings shed light on the involvement of ILC in COPD and provide insights into the potential interplay between elastin breakdown, immune cells, and disease progression. Further understanding of the mechanisms underlying ILC activation and their interaction with elastin peptides could contribute to the development of novel therapeutic strategies for COPD management.NEW & NOTEWORTHY Elastin-derived peptides, generated following alveolar degradation during emphysema in patients with COPD, are able to influence the response of type 2 innate lymphoid cells. We show that the orientation of innate lymphoid cells in patients with COPD is shifted toward a type 2 profile and that elastin peptides are indirectly participating in that shift through their influence of macrophages, which in turn impact innate lymphoid cells.

Membrane-based inverse-transition purification facilitates a rapid isolation of various spider-silk elastin-like polypeptide fusion proteins from extracts of transgenic tobacco.

Plants can produce complex pharmaceutical and technical proteins. Spider silk proteins are one example of the latter and can be used, for example, as compounds for high-performance textiles or wound dressings. If genetically fused to elastin-like polypeptides (ELPs), the silk proteins can be reversibly precipitated from clarified plant extracts at moderate temperatures of ~ 30 °C together with salt concentrations > 1.5 M, which simplifies purification and thus reduces costs. However, the technologies developed around this mechanism rely on a repeated cycling between soluble and aggregated state to remove plant host cell impurities, which increase process time and buffer consumption. Additionally, ELPs are difficult to detect using conventional staining methods, which hinders the analysis of unit operation performance and process development. Here, we have first developed a surface plasmon resonance (SPR) spectroscopy-based assay to quantity ELP fusion proteins. Then we tested different filters to prepare clarified plant extract with > 50% recovery of spider silk ELP fusion proteins. Finally, we established a membrane-based purification method that does not require cycling between soluble and aggregated ELP state but operates similar to an ultrafiltration/diafiltration device. Using a data-driven design of experiments (DoE) approach to characterize the system of reversible ELP precipitation we found that membranes with pore sizes up to 1.2 µm and concentrations of 2-3 M sodium chloride facilitate step a recovery close to 100% and purities of > 90%. The system can thus be useful for the purification of ELP-tagged proteins produced in plants and other hosts.

Design and Synthesis of Novel Ultralong-Acting Peptides as EDP-EBP Interaction Inhibitors for Pulmonary Fibrosis Treatment.

The increased remodeling of the extracellular matrix (ECM) in pulmonary fibrosis (PF) generates bioactive ECM fragments called matricryptins, which include elastin-derived peptides (EDPs). The interaction between EDPs and their receptors, including elastin-binding protein (EBP), plays a crucial role in exacerbating fibrosis. Here, we present LXJ-02 for the first time, a novel ultralong-acting inhibitor that disrupts the EDPs/EBP peptide-protein interaction, promoting macrophages to secrete matrix metalloproteinase-12 (MMP-12), and showing great promise as a stable peptide. MMP-12 has traditionally been implicated in promoting inflammation and fibrosis in various acute and chronic diseases. However, we reveal a novel role of LXJ-02 that activates the macrophage-MMP-12 axis to increase MMP-12 expression and degrade ECM components like elastin. This leads to the preventing of PF while also improving EDP-EBP interaction. LXJ-02 effectively reverses PF in mouse models with minimal side effects, holding great promise as an excellent therapeutic agent for lung fibrosis.

MFAP4-Deficiency Aggravates Age-Induced Changes in Resistance Artery Structure, While Ameliorating Hypertension.

BACKGROUND: Abnormalities of resistance arteries may play essential roles in the pathophysiology of aging and hypertension. Deficiency of the vascular extracellular matrix protein MFAP4 (microfibrillar-associated protein 4) has previously been observed as protective against aberrant arterial remodeling. We hypothesized that MFAP4-deficiency would reduce age- and hypertension-dependent arterial changes in extracellular matrix composition and stiffening. METHODS: Mesenteric arteries were isolated from old (20-23 months) littermate Mfap4+/+ and Mfap4-/- mice, and 2-photon excitation microscopy imaging was used to quantify elastin and collagen volumes and dimensions in the vascular wall. Ten-week-old littermate Mfap4+/+ and Mfap4-/- mice were subjected to 20 days of continuous Ang II (angiotensin II) infusion and hypertension was monitored using invasive blood pressure measurements. Arterial stiffness, responses to vascular constrictors, and myogenic tone were monitored using wire- or pressure-myography. Collagen contents were assessed by Western blotting. RESULTS: MFAP4-deficiency significantly increased collagen volume and elastin fragmentation in aged mesenteric arteries without affecting arterial stiffness. MFAP4-deficient mice exhibited reduced diastolic pressure in Ang II-induced hypertension. There was no significant effect of MFAP4-deficiency on mesenteric artery structural remodeling or myogenic tone, although collagen content in mesenteric arteries was tendentially increased in hypertensive Mfap4+/+ mice relative to Mfap4-/- mice. Increased efficacy of vasoconstrictors (phenylephrine, thromboxane) and reduced stiffness were observed in Ang II-treated Mfap4-/- mouse mesenteric arteries in ex vivo myography recordings. CONCLUSIONS: MFAP4-deficiency reduces the elastin/collagen ratio in the aging resistance artery without affecting arterial stiffness. In contrast, MFAP4-deficiency reduces the stiffness of resistance arteries and ameliorates Ang II-induced hypertension.

Morphological study of incompetent saphenous veins: apoptosis and ultrastructural changes of smooth muscle cells.

BACKGROUND: Varicose veins affect approximately 25% of people in industrialized countries. METHODS: The study aimed at detecting apoptotic cells and histopathological changes in varicose vein walls. Patients (N.=41) with varicose veins and 30 control group patients were divided into two groups according to their age (younger and older than 50 years). Apoptosis was determined by the TUNEL assay, elastin and collagen IV expression by immunohistochemistry and ultrastructural changes by transmission electron microscopy. RESULTS: The results show that the number of apoptotic cells in the layers of varicose veins increased, in particular in a group of patients aged over 50 years. In the varicose veins as compared to control veins the elastic fibers were found to be thinner, more fragmented and disorderly arranged. Elastin and collagen IV expression was found to decline in the intima and the media of varicose veins in both age groups. Electron microscopy demonstrated hypertrophy and degeneration of smooth muscle cells. Furthermore, cells with ultrastructural feature of apoptosis were noted. In the disorganized and expanded extracellular matrix membrane-bound vesicles, ghost bodies with different size and electron density were observed. Ghost bodies seem to bud off from smooth muscle cells and are likely to be involved in extracellular matrix remodeling as they are seen in close contact with collagen fibers. CONCLUSIONS: The study demonstrates increase of apoptotic cells in the wall of varicose veins along with vein wall structural abnormalities including alterations of smooth muscle cells and decline of elastin and collagen IV expression.

Extracellular Matrix Interactome in Modulating Vascular Homeostasis and Remodeling.

The ECM (extracellular matrix) is a major component of the vascular microenvironment that modulates vascular homeostasis. ECM proteins include collagens, elastin, noncollagen glycoproteins, and proteoglycans/glycosaminoglycans. ECM proteins form complex matrix structures, such as the basal lamina and collagen and elastin fibers, through direct interactions or lysyl oxidase-mediated cross-linking. Moreover, ECM proteins directly interact with cell surface receptors or extracellular secreted molecules, exerting matricellular and matricrine modulation, respectively. In addition, extracellular proteases degrade or cleave matrix proteins, thereby contributing to ECM turnover. These interactions constitute the ECM interactome network, which is essential for maintaining vascular homeostasis and preventing pathological vascular remodeling. The current review mainly focuses on endogenous matrix proteins in blood vessels and discusses the interaction of these matrix proteins with other ECM proteins, cell surface receptors, cytokines, complement and coagulation factors, and their potential roles in maintaining vascular homeostasis and preventing pathological remodeling.

Treatment with synchronized radiofrequency and facial muscle stimulation: Histologic analysis of human skin for changes in collagen and elastin fibers.

BACKGROUND: Skin's exposure to intrinsic and extrinsic factors causes age-related changes, leading to a lower amount of dermal collagen and elastin. AIM: This study investigated the effects of a novel facial muscle stimulation technology combined with radiofrequency (RF) heating on dermal collagen and elastin content for the treatment of facial wrinkles and skin laxity. METHODS: The active group subjects (N = 6) received four 20-min facial treatments with simultaneous RF and facial muscle stimulation, once weekly. The control subject (N = 1) was untreated. Skin biopsies obtained at baseline, 1-month and 3-month follow-up were evaluated histologically to determine collagen and elastin fibers content. A group of independent aestheticians evaluated facial skin appearance and wrinkle severity. Patient safety was followed. RESULTS: In the active group, collagen-occupied area reached 11.91 ± 1.80 × 106 µm2 (+25.32%, p < 0.05) and 12.35 ± 1.44 × 105 µm2 (+30.00%, p < 0.05) at 1-month and 3-month follow-up visits. Elastin-occupied area at 1-month and 3-month follow-up was 1.64 ± 0.14 × 105 µm2 (+67.23%, p < 0.05), and 1.99 ± 0.21 × 105 µm2 (+102.80%, p < 0.05). In the control group, there was no significant difference (p > 0.05) in collagen and elastin fibers. Active group wrinkle scores decreased from 5 (moderate, class II) to 3 (mild, class I). All subjects, except the control, improved in appearance posttreatment. No adverse events or side effects occurred. CONCLUSION: Decreased dermal collagen and elastin levels contributes to a gradual decline in skin elasticity, leading to facial wrinkles and unfirm skin. Study results showed noticeable improvement in facial appearance and increased dermal collagen and elastin content subsequent to simultaneous, noninvasive RF, and facial muscle stimulation treatments.

Comapping Cellular Content and Extracellular Matrix with Hemodynamics in Intact Arterial Tissues Using Scanning Immunofluorescent Multiphoton Microscopy.

Deviation of blood flow from an optimal range is known to be associated with the initiation and progression of vascular pathologies. Important open questions remain about how the abnormal flow drives specific wall changes in pathologies such as cerebral aneurysms where the flow is highly heterogeneous and complex. This knowledge gap precludes the clinical use of readily available flow data to predict outcomes and improve treatment of these diseases. As both flow and the pathological wall changes are spatially heterogeneous, a crucial requirement for progress in this area is a methodology for acquiring and comapping local vascular wall biology data with local hemodynamic data. Here, we developed an imaging pipeline to address this pressing need. A protocol that employs scanning multiphoton microscopy was developed to obtain three-dimensional (3D) datasets for smooth muscle actin, collagen, and elastin in intact vascular specimens. A cluster analysis was introduced to objectively categorize the smooth muscle cells (SMC) across the vascular specimen based on SMC actin density. Finally, direct quantitative comparison of local flow and wall biology in 3D intact specimens was achieved by comapping both heterogeneous SMC data and wall thickness to patient-specific hemodynamic results.

Rosemary extract and rosmarinic acid accelerate elastic fiber formation by increasing the expression of elastic fiber components in dermal fibroblasts.

Ultraviolet (UV)-induced skin photoaging is caused by qualitative and quantitative degradation of dermal extracellular matrix components such as collagen and elastic fibers. Elastic fibers are important for maintaining cutaneous elasticity, despite their small amount in the skin. Previously, microfibril-associated protein 4 (MFAP-4), which is downregulated in photoaging dermis, has been found to be essential for elastic fiber formation by interaction with both fibrillin-1 and elastin, which are core components of elastic fiber. In addition, enhanced cutaneous MFAP-4 expression in a human skin-xenografted murine photoaging model protects against UV-induced photodamage accompanied by the prevention of elastic fiber degradation and aggravated elasticity. We therefore hypothesized that the upregulation of MFAP-4 in dermal fibroblasts may more efficiently accelerate elastic fiber formation. We screened botanical extracts for MFAP-4 expression-promoting activity in normal human dermal fibroblasts (NHDFs). We found that rosemary extract markedly promotes early microfibril formation and mature elastic fiber formation along with a significant upregulation of not only MFAP-4 but also fibrillin-1 and elastin in NHDFs. Furthermore, rosmarinic acid, which is abundant in rosemary extract, accelerated elastic fiber formation via upregulation of transforming growth factor ß-1. This was achieved by the induction of cAMP response element-binding protein phosphorylation, demonstrating that rosmarinic acid represents one of the active ingredients in rosemary extract. Based on the findings in this study, we conclude that rosemary extract and rosmarinic acid represent promising materials that exert a preventive or ameliorative effect on skin photoaging by accelerating elastic fiber formation.

Elastin and collagen fibres in cutaneous wound healing.

Skin forms the outer barrier of the body. Upon injury, successful wound healing in normal skin restores tissue damage and counteracts the loss of extracellular matrix (ECM) proteins and cells. Collagens and elastin are the most abundant structural proteins of the ECM. In homeostasis, collagen type I is the prevalent form, but it is replaced by type III collagen upon wounding, and only later remodelled. In turn, unsuccessful healing results in scars, which tend to be inflexible and inelastic as compared to normal elastic dermis. Scar inelasticity may be due to the absence of mature elastin fibre formation and cross-linking. In this review, the available information on the process of formation of new collagen and elastic fibres during wound healing is analysed. The distinct roles of elastin and collagen proteins during healing are revisited and future research directions proposed which may help improve clinical management of open wounds and scars.

Novel high-yield potato protease inhibitor panels block a wide array of proteases involved in viral infection and crucial tissue damage.

Viruses critically rely on various proteases to ensure host cell entry and replication. In response to viral infection, the host will induce acute tissue inflammation pulled by granulocytes. Upon hyperactivation, neutrophil granulocytes may cause undue tissue damage through proteolytic degradation of the extracellular matrix. Here, we assess the potential of protease inhibitors (PI) derived from potatoes in inhibiting viral infection and reducing tissue damage. The original full spectrum of potato PI was developed into five fractions by means of chromatography and hydrolysis. Individual fractions showed varying inhibitory efficacy towards a panel of proteases including trypsin, chymotrypsin, ACE2, elastase, and cathepsins B and L. The fractions did not interfere with SARS-CoV-2 infection of Vero E6 cells in vitro. Importantly, two of the fractions fully inhibited elastin-degrading activity of complete primary human neutrophil degranulate. These data warrant further development of potato PI fractions for biomedical purposes, including tissue damage crucial to SARS-CoV-2 pathogenesis. KEY MESSAGES: Protease inhibitor fractions from potato differentially inhibit a series of human proteases involved in viral replication and in tissue damage by overshoot inflammation. Protease inhibition of cell surface receptors such as ACE2 does not prevent virus infection of Vero cells in vitro. Protease inhibitors derived from potato can fully inhibit elastin-degrading primary human neutrophil proteases. Protease inhibitor fractions can be produced at high scale (hundreds of thousands of kilograms, i.e., tons) allowing economically feasible application in lower and higher income countries.

Uniaxial mechanical stretch properties correlated with three-dimensional microstructure of human dermal skin.

The intact and healthy skin forms a barrier to the outside world and protects the body from mechanical impact. The skin is a complex structure with unique mechano-elastic properties. To better direct the design of biomimetic materials and induce skin regeneration in wounds with optimal outcome, more insight is required in how the mechano-elastic properties emerge from the skin's main constituents, collagen and elastin fibers. Here, we employed two-photon excited autofluorescence and second harmonic generation microscopy to characterize collagen and elastin fibers in 3D in 24 human dermis skin samples. Through uniaxial stretching experiments, we derive uni-directional mechanical properties from resultant stress-strain curves, including the initial Young's modulus, elastic Young's modulus, maximal stress, and maximal and mid-strain values. The stress-strain curves show a large variation, with an average Young's modules in the toe and linear regions of 0.1 MPa and 21 MPa. We performed a comprehensive analysis of the correlation between the key mechanical properties with age and with microstructural parameters, e.g., fiber density, thickness, and orientation. Age was found to correlate negatively with Young's modulus and collagen density. Moreover, real-time monitoring during uniaxial stretching allowed us to observe changes in collagen and elastin alignment. Elastin fibers aligned significantly in both the heel and linear regions, and the collagen bundles engaged and oriented mainly in the linear region. This research advances our understanding of skin biomechanics and yields input for future first principles full modeling of skin tissue.

Vitamin C, lactoferrin and elastin-Advancing the science.

BACKGROUND: This study follows an initial scientific validation linking sodium ascorbate (SAC) with elastin conservation and the clinical trial histology observation that the full formulation tested there stimulated elastin development. In an effort to explain the increased elastin response, a candidate was sought that may provide synergy to SAC during elastin stimulation. Lactoferrin was the constituent chosen to explore in this realm. MATERIALS AND METHODS: Using the previously described ex vivo skin model, freshly collected discarded human skin from 2 donors was used to evaluate the effects of lactoferrin and SAC alone and together, and L-ascorbate CE Ferulic formulation (CEF) on elastogenesis. Four skin explants were topically subjected to the treatments daily for 7 days and one group was left untreated as a negative control. The tissue was fixed and embedded. Sections were evaluated by immunofluorescence using antibodies targeting Tropoelastin and CD44, with DAPI counterstaining to observe nuclei. Images were then analyzed using ImageJ. RESULTS: Treatment with SAC and lactoferrin demonstrated a significant synergistic effect on tropoelastin stimulation compared to the single treatments. In addition, this combination demonstrated intact and increased elastin fibers in contrast to the CEF, which portrayed fragmented elastin fibers. In addition, an additive effect of SAC also contributed to the enhanced CD44, suggesting an increased presence of hyaluronic acid, a new observation for this compound. CONCLUSION: This study complements a series of studies that have been undertaken to validate the efficacy of a novel antioxidant formulation. Aside from its efficacy in ROS management, the SAC constituent is unique in the different forms of Vitamin C for its ability to conserve elastin. Prior clinical studies demonstrated additive elastin stimulation on histology, not just conservation. From this current study, the combination of SAC with lactoferrin may be responsible for this additive stimulatory effect on elastin. This presents a significant advance in topical antioxidant formulations where the Vitamin C component provides antioxidant and collagen stimulation with additional elastin stimulation rather than degradation.

Sialic acids cleavage induced by elastin-derived peptides impairs the interaction between insulin and its receptor in adipocytes 3T3-L1.

The insulin receptor (IR) plays an important role in insulin signal transduction, the defect of which is believed to be the root cause of type 2 diabetes. In 3T3-L1 adipocytes as in other cell types, the mature IR is a heterotetrameric cell surface glycoprotein composed of two α subunits and two ß subunits. Our objective in our study, is to understand how the desialylation of N-glycan chains, induced by elastin-derived peptides, plays a major role in the function of the IR. Using the 3T3-L1 adipocyte line, we show that removal of the sialic acid from N-glycan chains (N893 and N908), induced by the elastin receptor complex (ERC) and elastin derived-peptides (EDPs), leads to a decrease in the autophosphorylation activity of the insulin receptor. We demonstrate by molecular dynamics approaches that the absence of sialic acids on one of these two sites is sufficient to generate local and general modifications of the structure of the IR. Biochemical approaches highlight a decrease in the interaction between insulin and its receptor when ERC sialidase activity is induced by EDPs. Therefore, desialylation by EDPs is synonymous with a decrease of IR sensitivity in adipocytes and could thus be a potential source of insulin resistance associated with diabetic conditions.

Contributions of collagen and elastin to elastic behaviours of tendon fascicle.

Tendon exhibits the capacity to be stretched and to return to its original length without suffering structural damage in vivo, a capacity known as elastic recoil. Collagen fibres are aligned longitudinally and elastin fibres mostly run parallel to collagen fibres in tendon. However, their interactions and contributions to tendon elastic behaviours are not well understood. The present study examined functional roles of collagen and elastin in tendon elastic behaviours using a variety of mechanical tests. We prepared three types of fascicle specimens from mouse tail tendon: fascicles freshly isolated, those digested with elastase in PBS to selectively remove elastin, and those incubated in PBS without elastase. A quasi-static tensile test demonstrated that elastase-treated fascicles had higher tangent moduli and strength compared to fresh and PBS fascicles. Cyclic stretching tests showed that fresh and PBS fascicles could withstand cyclic strain at both small and large amplitudes, but elastase-treated fascicles could only behave elastically to a limited degree. Fibre-sliding analysis revealed that fresh fascicles could be elongated both through stretching of collagen fibers and through movement of the fibres. However, elastase-treated fascicles could be stretched only via fibre stretching. This evidence suggests that normal tendons can be extended through both fibre stretching and fibre sliding, whereas tendons without elastin can only extend as much as collagen fibers can withstand. Accordingly, collagen fibres mainly contribute to tendon elastic behaviours by furnishing rigidity and elasticity, whereas elastin provides tendon viscoelasticity and also enables sliding of collagen fibres during elastic behaviours. STATEMENT OF SIGNIFICANCE: The present study revealed distinct mechanical functions of collagen and elastin fibres in elastic behaviours of mouse tail tendon fascicle using a variety of mechanical tests at both microscopic and macroscopic levels. It was demonstrated that collagen mainly governs tendon fascicle rigidity and elasticity, but only possesses limited extensibility, whereas elastin contributes to viscoelasticity and collagen fibre sliding, enabling elastic recoil behaviour against relatively large deformation. By their interactions, tendon can be elongated without suffering major structural damage and withstand a large magnitude of tensile force in response to mechanical loading. Such information should be particularly useful in designing collagen-based biomaterials such as artificial tendons, in that previous studies have merely considered collagen without incorporation of elastin.

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.

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.