Elastin recoil is driven by the hydrophobic effect.

Elastin is an extracellular matrix material found in all vertebrates. Its reversible elasticity, robustness, and low stiffness are essential for the function of arteries, lungs, and skin. It is among the most resilient elastic materials known: During a human lifetime, arterial elastin undergoes in excess of 2 × 109 stretching/contracting cycles without replacement, and slow oxidative hardening has been identified as a limiting factor on human lifespan. For over 50 y, the mechanism of entropic recoil has been controversial. Herein, we report a combined NMR and thermomechanical study that establishes the hydrophobic effect as the primary driver of elastin function. Water ordering at the solvent:protein interface was observed as a function of stretch using double quantum 2H NMR, and the most extensive thermodynamic analysis performed to date was obtained by measuring elastin length and volume as a function of force and temperature in normal water, heavy water and with cosolvents. When stretched, elastin's heat capacity increases, water is ordered proportional to the degree of stretching, the internal energy decreases, and heat is released in excess of the work performed. These properties show that recoil in elastin under physiological conditions is primarily driven by the hydrophobic effect rather than by configurational entropy as is the case for rubber. Consistent with this conclusion are decreases in the thermodynamic signatures when cosolvents that alter the hydrophobic effect are introduced. We propose that hydrophobic effect-driven recoil, as opposed to a configurational entropy mechanism where hardening from crystallization can occur, is the origin of elastin's unusual resilience.

In situ formed depot of elastin-like polypeptide-hirudin fusion protein for long-acting antithrombotic therapy.

Thrombosis, induced by abnormal coagulation or fibrinolytic systems, is the most common pathology associated with many life-threatening cardio-cerebrovascular diseases. However, first-line anticoagulant drugs suffer from rapid drug elimination and risk of hemorrhagic complications. Here, we developed an in situ formed depot of elastin-like polypeptide (ELP)-hirudin fusion protein with a prodrug-like feature for long-term antithrombotic therapy. Highly secretory expression of the fusion protein was achieved with the assistance of the Ffu312 tag. Integration of hirudin, ELP, and responsive moiety can customize fusion proteins with properties of adjustable in vivo retention and controllable recovery of drug bioactivity. After subcutaneous injection, the fusion protein can form a reservoir through temperature-induced coacervation of ELP and slowly diffuse into the blood circulation. The biological activity of hirudin is shielded due to the N-terminal modification, while the activated key proteases upon thrombus occurrence trigger the cleavage of fusion protein together with the release of hirudin, which has antithrombotic activity to counteract thrombosis. We substantiated that the optimized fusion protein produced long-term antithrombotic effects without the risk of bleeding in multiple animal thrombosis models.

Structural and physical basis for the elasticity of elastin.

Elastin function is to endow vertebrate tissues with elasticity so that they can adapt to local mechanical constraints. The hydrophobicity and insolubility of the mature elastin polymer have hampered studies of its molecular organisation and structure-elasticity relationships. Nevertheless, a growing number of studies from a broad range of disciplines have provided invaluable insights, and several structural models of elastin have been proposed. However, many questions remain regarding how the primary sequence of elastin (and the soluble precursor tropoelastin) governs the molecular structure, its organisation into a polymeric network, and the mechanical properties of the resulting material. The elasticity of elastin is known to be largely entropic in origin, a property that is understood to arise from both its disordered molecular structure and its hydrophobic character. Despite a high degree of hydrophobicity, elastin does not form compact, water-excluding domains and remains highly disordered. However, elastin contains both stable and labile secondary structure elements. Current models of elastin structure and function are drawn from data collected on tropoelastin and on elastin-like peptides (ELPs) but at the tissue level, elasticity is only achieved after polymerisation of the mature elastin. In tissues, the reticulation of tropoelastin chains in water defines the polymer elastin that bears elasticity. Similarly, ELPs require polymerisation to become elastic. There is considerable interest in elastin especially in the biomaterials and cosmetic fields where ELPs are widely used. This review aims to provide an up-to-date survey of/perspective on current knowledge about the interplay between elastin structure, solvation, and entropic elasticity.

The Genetics and Typical Traits of Thoracic Aortic Aneurysm and Dissection.

Genetic predisposition and risk factors such as hypertension and smoking can instigate the development of thoracic aortic aneurysm (TAA), which can lead to highly lethal aortic wall dissection and/or rupture. Monogenic defects in multiple genes involved in the elastin-contractile unit and the TGFß signaling pathway have been associated with TAA in recent years, along with several genetic modifiers and risk-conferring polymorphisms. Advances in omics technology have also provided significant insights into the processes behind aortic wall degeneration: inflammation, epigenetics, vascular smooth muscle phenotype change and depletion, reactive oxygen species generation, mitochondrial dysfunction, and angiotensin signaling dysregulation. These recent advances and findings might pave the way for a therapy that is capable of stopping and perhaps even reversing aneurysm progression.

Tropoelastin Improves Post-Infarct Cardiac Function.

BACKGROUND: Myocardial infarction (MI) is among the leading causes of death worldwide. Following MI, necrotic cardiomyocytes are replaced by a stiff collagen-rich scar. Compared to collagen, the extracellular matrix protein elastin has high elasticity and may have more favorable properties within the cardiac scar. We sought to improve post-MI healing by introducing tropoelastin, the soluble subunit of elastin, to alter scar mechanics early after MI. METHODS AND RESULTS: We developed an ultrasound-guided direct intramyocardial injection method to administer tropoelastin directly into the left ventricular anterior wall of rats subjected to induced MI. Experimental groups included shams and infarcted rats injected with either PBS vehicle control or tropoelastin. Compared to vehicle treated controls, echocardiography assessments showed tropoelastin significantly improved left ventricular ejection fraction (64.7±4.4% versus 46.0±3.1% control) and reduced left ventricular dyssynchrony (11.4±3.5 ms versus 31.1±5.8 ms control) 28 days post-MI. Additionally, tropoelastin reduced post-MI scar size (8.9±1.5% versus 20.9±2.7% control) and increased scar elastin (22±5.8% versus 6.2±1.5% control) as determined by histological assessments. RNA sequencing (RNAseq) analyses of rat infarcts showed that tropoelastin injection increased genes associated with elastic fiber formation 7 days post-MI and reduced genes associated with immune response 11 days post-MI. To show translational relevance, we performed immunohistochemical analyses on human ischemic heart disease cardiac samples and showed an increase in tropoelastin within fibrotic areas. Using RNA-seq we also demonstrated the tropoelastin gene ELN is upregulated in human ischemic heart disease and during human cardiac fibroblast-myofibroblast differentiation. Furthermore, we showed by immunocytochemistry that human cardiac fibroblast synthesize increased elastin in direct response to tropoelastin treatment. CONCLUSIONS: We demonstrate for the first time that purified human tropoelastin can significantly repair the infarcted heart in a rodent model of MI and that human cardiac fibroblast synthesize elastin. Since human cardiac fibroblasts are primarily responsible for post-MI scar synthesis, our findings suggest exciting future clinical translation options designed to therapeutically manipulate this synthesis.

De Novo Elastin Assembly Alleviates Development of Supravalvular Aortic Stenosis-Brief Report.

BACKGROUND: A series of incurable cardiovascular disorders arise due to improper formation of elastin during development. Supravalvular aortic stenosis (SVAS), resulting from a haploinsufficiency of ELN, is caused by improper stress sensing by medial vascular smooth muscle cells, leading to progressive luminal occlusion and heart failure. SVAS remains incurable, as current therapies do not address the root issue of defective elastin. METHODS: We use SVAS here as a model of vascular proliferative disease using both human induced pluripotent stem cell-derived vascular smooth muscle cells and developmental Eln+/- mouse models to establish de novo elastin assembly as a new therapeutic intervention. RESULTS: We demonstrate mitigation of vascular proliferative abnormalities following de novo extracellular elastin assembly through the addition of the polyphenol epigallocatechin gallate to SVAS human induced pluripotent stem cell-derived vascular smooth muscle cells and in utero to Eln+/- mice. CONCLUSIONS: We demonstrate de novo elastin deposition normalizes SVAS human induced pluripotent stem cell-derived vascular smooth muscle cell hyperproliferation and rescues hypertension and aortic mechanics in Eln+/- mice, providing critical preclinical findings for the future application of epigallocatechin gallate treatment in humans.

Accelerating Proteomics Using Broad Specificity Proteases.

Trypsin is the gold-standard protease in bottom-up proteomics, but many sequence stretches of the proteome are inaccessible to trypsin and standard LC-MS approaches. Thus, multienzyme strategies are used to maximize sequence coverage in post-translational modification profiling. We present fast and robust SP3- and STRAP-based protocols for the broad-specificity proteases subtilisin, proteinase K, and thermolysin. All three enzymes are remarkably fast, producing near-complete digests in 1-5 min, and cost 200-1000× less than proteomics-grade trypsin. Using FragPipe resolved a major challenge by drastically reducing the duration of the required "unspecific" searches. In-depth analyses of proteinase K, subtilisin, and thermolysin Jurkat digests identified 7374, 8178, and 8753 unique proteins with average sequence coverages of 21, 29, and 37%, including 10,000s of amino acids not reported in PeptideAtlas' >2400 experiments. While we could not identify distinct cleavage patterns, machine learning could distinguish true protease products from random cleavages, potentially enabling the prediction of cleavage products. Finally, proteinase K, subtilisin, and thermolysin enabled label-free quantitation of 3111, 3659, and 4196 unique Jurkat proteins, which in our hands is comparable to trypsin. Our data demonstrate that broad-specificity proteases enable quantitative proteomics of uncharted areas of the proteome. Their fast kinetics may allow "on-the-fly" digestion of samples in the future.

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.

Citrullination, a novel post-translational modification of elastin, is involved in COPD Pathogenesis.

Elastin is an extracellular matrix protein (ECM) that supports elasticity of the lung, and in patients with chronic obstructive pulmonary disease (COPD) and emphysema, the structural changes that reduce the amount of elastic recoil, lead to loss of pulmonary function. We recently demonstrated that elastin is a target of peptidyl arginine deiminase (PAD) enzyme-induced citrullination, thereby leading to enhanced susceptibility of this ECM protein to proteolysis. The current study aimed to investigate the impact of PAD activity in vivo and furthermore assessed whether pharmacological inhibition of PAD activity protects against pulmonary emphysema. Using a Serpina1a-e knockout mouse model, previously shown to develop inflammation-mediated emphysema, we validated the involvement of PADs in airway disease. In line with emphysema development, intratracheal administration of lipopolysaccharide in combination with PADs provoked significant airspace enlargement (P < 0.001) and diminished lung function, including loss of lung tissue elastance (P = 0.0217) and increases in lung volumes (P = 0.0463). Intraperitoneal treatment of mice with the PAD inhibitor, BB-Cl-amidine, prevented PAD/LPS-mediated lung function decline and emphysema and reduced levels of citrullinated airway elastin (P = 0.0199). These results provide evidence for the impact of PADs on lung function decline, indicating promising potential for the future development of PAD-based therapeutics for preserving lung function in patients with COPD.

Accumulation of extracellular elastin-derived peptides disturbed neuronal morphology and neuron-microglia crosstalk in aged brain.

Extracellular elastin-derived peptides (EDPs) accumulate in the aging brain and have been associated with vascular dementia and Alzheimer's disease (AD). The activation of inflammatory processes in glial cells with EDP treatment has received attention, but not in neurons. To properly understand EDPs' pathogenic significance, the impact on neuronal function and neuron-microglia crosstalk was explored further. Among the EDP molecules, Val-Gly-Val-Ala-Pro-Gly (VGVAPG) is a typical repeating hexapeptide. Here, we observed that EDPs-VGVAPG influenced neuronal survival and morphology in a dose-dependent manner. High concentrations of VGVAPG induced synapse loss and microglia hyperactivation in vivo and in vitro. Following EDP incubation, galectin 3 (Gal-3) released by neurons served as a chemokine, attracting microglial engulfment. Blocking Gal-3 and EDP binding remedied synapse loss in neurons and phagocytosis in microglia. In response to the accumulation of EDPs, proteomics in matrix remodeling and cytoskeleton dynamics, such as a disintegrin and metalloproteinase (ADAM) family, were engaged. These findings in extracellular EDPs provided more evidence for the relationship between aging and neuron dysfunction, increasing the insight of neuroinflammatory responses and the development of new specialized extracellular matrix remolding-targeted therapy options for dementia or other neurodegenerative disease.

Protective Effect of Red Light-Emitting Diode against UV-B Radiation-Induced Skin Damage in SKH:HR-2 Hairless Mice.

In this in vivo study on hairless mice, we examined the effects of light-emitting diode (LED) treatment applied prior to ultraviolet B (UVB) irradiation. We found that pre-treating with LED improved skin morphological and histopathological conditions compared to those only exposed to UVB irradiation. In our study, histological evaluation of collagen and elastic fibers after LED treatment prior to UVB irradiation showed that this pretreatment significantly enhanced the quality of fibers, which were otherwise poor in density and irregularly arranged due to UV exposure alone. This suggests that LED treatment promotes collagen and elastin production, leading to improved skin properties. Additionally, we observed an increase in Claudin-1 expression and a reduction in nuclear factor-erythroid 2-related factor 2 (Nrf-2) and heme-oxygenase 1 (HO-1) expression within the LED-treated skin tissues, suggesting that LED therapy may modulate key skin barrier proteins and oxidative stress markers. These results demonstrate that pretreatment with LED light can enhance the skin's resistance to UVB-induced damage by modulating gene regulation associated with skin protection. Further investigations are needed to explore the broader biological effects of LED therapy on other tissues such as blood vessels. This study underscores the potential of LED therapy as a non-invasive approach to enhance skin repair and counteract the effects of photoaging caused by UV exposure.

Greater TIMP-1 protein levels and neointimal formation represent sex-dependent cellular events limiting aortic vessel expansion in female rats.

Fragmentation/loss of the structural protein elastin represents the precipitating event translating to aortic expansion and subsequent aneurysm formation. The present study tested the hypothesis that greater protein expression of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and neointimal growth secondary to a reduction of medial elastin content represent sex-dependent events limiting aortic vessel expansion in females. TIMP-1 protein levels were higher in the ascending aorta of female versus male patients diagnosed with a bicuspid aortic valve (BAV). The latter paradigm was recapitulated in the aorta of adult male and female rats complemented by greater TIMP-2 expression in females. CaCl2 (0.5 M) treatment of the infrarenal aorta of adult male and female rats increased the in situ vessel diameter and expansion was significantly smaller in females despite a comparable reduction of medial elastin content. The preferential appearance of a neointimal region of the CaCl2-treated infrarenal aorta of female rats may explain in part the smaller in situ expansion and neointimal growth correlated positively with the % change of the in situ diameter. Neointimal formation was secondary to a significant increase in the density of medial/neointimal vascular smooth muscle cells (VSMCs) that re-entered the G2-M phase whereas VSMC cell cycle re-entry was attenuated in the CaCl2-treated infrarenal aorta of male rats. Thus, greater TIMP-1 expression in the aorta of female BAV patients may prevent excessive elastin fragmentation and preferential neointimal growth following CaCl2-treatment of the infrarenal aorta of female rats represents a sex-dependent biological event limiting vessel expansion secondary to a significant loss of the structural protein.

Elastin-derived peptides (EDPs) affect gene and protein expression in human mesenchymal stem cells (hMSCs) - preliminary study.

During the aging process, elastin is degraded and the level of elastin-derived peptides (EDPs) successively increases. The main peptide released from elastin during its degradation is a peptide with the VGVAPG sequence. To date, several papers have described that EDPs or elastin-like peptides (ELPs) affect human mesenchymal stem cells (hMSCs) derived from different tissues. Unfortunately, despite the described effect of EDPs or ELPs on the hMSC differentiation process, the mechanism of action of these peptides has not been elucidated. Therefore, the aim of the present study was to evaluate the impact of the VGVAPG and VVGPGA peptides on the hMSC stemness marker and elucidation of the mechanism of action of these peptides. Our data show that both studied peptides (VGVAPG and VVGPGA) act with the involvement of ERK1/2 and c-SRC kinases. However, their mechanism of activation is probably different in hMSCs derived from adipose tissue. Both studied peptides increase the KI67 protein level in hMSCs, but this is not accompanied with cell proliferation. Moreover, the changes in the NANOG and c-MYC protein expression and in the SOX2 and POU5F1 mRNA expression suggest that EDPs reduced the hMSC stemness properties and could initiate cell differentiation. The initiation of differentiation was evidenced by changes in the expression of AhR and PPARγ protein as well as specific genes (ACTB, TUBB3) and proteins (ß-actin, RhoA) involved in cytoskeleton remodeling. Our data suggest that the presence of EDPs in tissue can initiate hMSC differentiation into more tissue-specific cells.

Lysyl oxidase like-1 deficiency in optic nerve head astrocytes elicits reactive astrocytosis and alters functional effects of astrocyte derived exosomes.

Glaucoma is a multifactorial progressive ocular pathology that manifests clinically with damage to the optic nerve (ON) and the retina, ultimately leading to blindness. The optic nerve head (ONH) shows the earliest signs of glaucoma pathology, and therefore, is an attractive target for drug discovery. The goal of this study was to elucidate the effects of reactive astrocytosis on the elastin metabolism pathway in primary rat optic nerve head astrocytes (ONHA), the primary glial cell type in the unmyelinated ONH. Following exposure to static equibiaxial mechanical strain, we observed prototypic molecular and biochemical signatures of reactive astrocytosis that were associated with a decrease in lysyl oxidase like 1 (Loxl1) expression and a concomitant decrease in elastin (Eln) gene expression. We subsequently investigated the role of Loxl1 in reactive astrocytosis by generating primary rat ONHA cultures with ∼50% decreased Loxl1 expression. Our results suggest that reduced Loxl1 expression is sufficient to elicit molecular signatures of elastinopathy in ONHA. Astrocyte derived exosomes (ADE) significantly increased the length of primary neurites of primary neurons in vitro. In contrast, ADE from Loxl1-deficient ONHA were deficient of trophic effects on neurite outgrowth in vitro, positing that Loxl1 dysfunction and the ensuing impaired elastin synthesis during reactive astrocytosis in the ONH may contribute to impaired neuron-glia signaling in glaucoma. Our data support a role of dysregulated Loxl1 function in eliciting reactive astrocytosis in glaucoma subtypes associated with increased IOP, even in the absence of genetic polymorphisms in LOXL1 typically associated with exfoliation glaucoma. This suggests the need for a paradigm shift toward considering lysyl oxidase activity and elastin metabolism and signaling as contributors to an altered secretome of the ONH that may lead to the progression of glaucomatous changes. Future research is needed to investigate cargo of exosomes in the context of reactive astrocytosis and identify the pathways leading to the observed transcriptome changes during reactive astrocytosis.

Elastin-Like Peptide as a Model for Disordered Proteins: Diffusion Behaviour in Self-Crowding Conditions.

Despite the current high interest, there is limited information on diffusion data for intrinsically disordered proteins (IDPs). This study investigates the effect of crowding on the diffusion behaviour of an elastin-like peptide (ELP), by combined pulse field gradient (PFG) and static field gradient (SFG) NMR techniques. We interpret our findings in terms of highly dynamic chain assemblies with weak interactions, resulting in ELP diffusion that is primarily governed by the viscous flow of the solvent. The diffusion behaviour of the peptide appears to resemble that of globular proteins rather than flexible linear polymers over a wide concentration range.

Improved self-cleaving precipitation tags for efficient column free bioseparations.

Current biological research requires simple protein bioseparation methods capable of purifying target proteins in a single step with high yields and purities. Conventional affinity tag-based approaches require specific affinity resins and expensive proteolytic enzymes for tag removal. Purification strategies based on self-cleaving aggregating tags have been previously developed to address these problems. However, these methods often utilize C-terminal cleaving contiguous inteins which suffer from premature cleavage, resulting in significant product loss during protein expression. In this work, we evaluate two novel mutants of the Mtu RecA ΔI-CM mini-intein obtained through yeast surface display for improved protein purification. When used with the elastin-like-polypeptide (ELP) precipitation tag, the novel mutants - ΔI-12 and ΔI-29 resulted in significantly higher precursor content, product purity and process yield compared to the original Mtu RecA ΔI-CM mini-intein. Product purities ranging from 68 % to 94 % were obtained in a single step for three model proteins - green fluorescent protein (GFP), maltose binding protein (MBP) and beta-galactosidase (beta-gal). Further, high cleaving efficiency was achieved after 5 h under most conditions. Overall, we have developed improved self-cleaving precipitation tags which can be used for purifying a wide range of proteins cheaply at laboratory scale.

Unlocking the potential of Extensin Signal peptide and Elastin-like polypeptide tag fused to Shigella dysenteriae's IpaDSTxB to improve protein expression and purification in Nicotiana tabacum and Medicagosativa.

Plants are often seen as a potent tool in the recombinant protein production industry. However, unlike bacterial expression, it is not a popular method due to the low yield and difficulty of protein extraction and purification. Therefore, developing a new high efficient and easy to purify platform is crucial. One of the best approaches to make extraction easier is to utilize the Extensin Signal peptide (EXT) to translocate the recombinant protein to the outside of the cell, along with incorporating an Elastin-like polypeptide tag (ELP) to enhance purification and accumulation rates. In this research, we transiently expressed Shigella dysenteriae's IpaDSTxB fused to both NtEXT and ELP in both Nicotiana tabacum and Medicago sativa. Our results demonstrated that N. tabacum, with an average yield of 6.39 ng/µg TSP, outperforms M. sativa, which had an average yield of 3.58 ng/µg TSP. On the other hand, analyzing NtEXT signal peptide indicated that merging EXT to the constructs facilitates translocation of IpaDSTxB to the apoplast by 78.4% and 65.9% in N. tabacum and M. sativa, respectively. Conversely, the mean level for constructs without EXT was below 25% for both plants. Furthermore, investigation into the orientation of ELP showed that merging it to the C-terminal of IpaDSTxB leads to a higher accumulation rate in both N. tabacum and M. sativa by 1.39 and 1.28 times, respectively. It also facilitates purification rate by over 70% in comparison to 20% of the 6His tag. The results show a highly efficient and easy to purify platform for the expression of heterologous proteins in plant.

Development of an Elastin-like Polypeptide-Based Nucleic Acid Delivery System Targeted to EGFR+ Bladder Cancer Cells Using a Layer-by-Layer Approach.

Nucleic acid (NA)-based therapies are revolutionizing biomedical research through their ability to control cellular functions at the genetic level. This work demonstrates a versatile elastin-like polypeptide (ELP) carrier system using a layer-by-layer (LbL) formulation approach that delivers NA cargos ranging in size from siRNA to plasmids. The components of the system can be reconfigured to modulate the biochemical and biophysical characteristics of the carrier for engaging the unique features of the biological target. We show the physical characterization and biological performance of LbL ELP nucleic acid nanoparticles (LENNs) in murine and human bladder tumor cell lines. Targeting bladder tumors is difficult owing to the constant influx of urine into the bladder, leading to low contact times (typically <2 h) for therapeutic agents delivered via intravesical instillation. LENN complexes bind to bladder tumor cells within 30 min and become rapidly internalized to release their NA cargo within 60 min. Our data show that a readily adaptable NA-delivery system has been created that is flexible in its targeting ability, cargo size, and disassembly kinetics. This approach provides an alternative path to either lipid nanoparticle formulations that suffer from inefficiency and physicochemical instability or viral vectors that are plagued by manufacturing and immune rejection challenges. This agile ELP-based nanocarrier provides an alternative route for nucleic acid delivery using a biomanufacturable, biodegradable, biocompatible, and highly tunable vehicle capable of targeting cells via engagement with overexpressed cell surface receptors.

Association of NOTCH3 With Elastic Fiber Dispersion in the Infrarenal Abdominal Aorta of Cynomolgus Monkeys.

BACKGROUND: The regional heterogeneity of vascular components and transcriptomes is an important determinant of aortic biology. This notion has been explored in multiple mouse studies. In the present study, we examined the regional heterogeneity of aortas in nonhuman primates. METHODS: Aortic samples were harvested from the ascending, descending thoracic, suprarenal, and infrarenal regions of young control monkeys and adult monkeys with high fructose consumption for 3 years. The regional heterogeneity of aortic structure and transcriptomes was examined by histological and bulk RNA sequencing analyses, respectively. RESULTS: Immunostaining of CD31 and αSMA (alpha-smooth muscle actin) revealed that endothelial and smooth muscle cells were distributed homogeneously across the aortic regions. In contrast, elastic fibers were less abundant and dispersed in the infrarenal aorta compared with other regions and associated with collagen deposition. Bulk RNA sequencing identified a distinct transcriptome related to the Notch signaling pathway in the infrarenal aorta with significantly increased NOTCH3 mRNA compared with other regions. Immunostaining revealed that NOTCH3 protein was increased in the media of the infrarenal aorta. The abundance of medial NOTCH3 was positively correlated with the dispersion of elastic fibers. Adult cynomolgus monkeys with high fructose consumption displayed vascular wall remodeling, such as smooth muscle cell loss and elastic fiber disruption, predominantly in the infrarenal region. The correlation between NOTCH3 and elastic fiber dispersion was enhanced in these monkeys. CONCLUSIONS: Aortas of young cynomolgus monkeys display regional heterogeneity of their transcriptome and the structure of elastin and collagens. Elastic fibers in the infrarenal aorta are dispersed along with upregulation of medial NOTCH3.

DOCK2 Deficiency Attenuates Abdominal Aortic Aneurysm Formation-Brief Report.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a potentially lethal disease that lacks pharmacological treatment. Degradation of extracellular matrix proteins, especially elastin laminae, is the hallmark for AAA development. DOCK2 (dedicator of cytokinesis 2) has shown proinflammatory effects in several inflammatory diseases and acts as a novel mediator for vascular remodeling. However, the role of DOCK2 in AAA formation remains unknown. METHODS: Ang II (angiotensin II) infusion of ApoE-/- (apolipoprotein E deficient) mouse and topical elastase-induced AAA combined with DOCK2-/- (DOCK2 knockout) mouse models were used to study DOCK2 function in AAA formation/dissection. The relevance of DOCK2 to human AAA was examined using human aneurysm specimens. Elastin fragmentation in AAA lesion was observed by elastin staining. Elastin-degrading enzyme MMP (matrix metalloproteinase) activity was measured by in situ zymography. RESULTS: DOCK2 was robustly upregulated in AAA lesion of Ang II-infused ApoE-/- mice, elastase-treated mice, as well as human AAA lesions. DOCK2-/- significantly attenuated the Ang II-induced AAA formation/dissection or rupture in mice along with reduction of MCP-1 (monocyte chemoattractant protein-1) and MMP expression and activity. Accordingly, the elastin fragmentation observed in ApoE-/- mouse aorta infused with Ang II and elastase-treated aorta was significantly attenuated by DOCK2 deficiency. Moreover, DOCK2-/- decreased the prevalence and severity of aneurysm formation, as well as the elastin degradation observed in the topical elastase model. CONCLUSIONS: Our results indicate that DOCK2 is a novel regulator for AAA formation. DOCK2 regulates AAA development by promoting MCP-1 and MMP2 expression to incite vascular inflammation and elastin degradation.