Nanoparticle-based targeted delivery of pentagalloyl glucose reverses elastase-induced abdominal aortic aneurysm and restores aorta to the healthy state in mice.

AIM: Abdominal aortic aneurysms (AAA) is a life-threatening weakening and expansion of the abdominal aorta due to inflammatory cell infiltration and gradual degeneration of extracellular matrix (ECM). There are no pharmacological therapies to treat AAA. We tested the hypothesis that nanoparticle (NP) therapy that targets degraded elastin and delivers anti-inflammatory, anti-oxidative, and ECM stabilizing agent, pentagalloyl glucose (PGG) will reverse advance stage aneurysm in an elastase-induced mouse model of AAA. METHOD AND RESULTS: Porcine pancreatic elastase (PPE) was applied periadventitially to the infrarenal aorta in mice and AAA was allowed to develop for 14 days. Nanoparticles loaded with PGG (EL-PGG-NPs) were then delivered via IV route at 14-day and 21-day (10 mg/kg of body weight). A control group of mice received no therapy. The targeting of NPs to the AAA site was confirmed with fluorescent dye marked NPs and gold NPs. Animals were sacrificed at 28-d. We found that targeted PGG therapy reversed the AAA by decreasing matrix metalloproteinases MMP-9 and MMP-2, and the infiltration of macrophages in the medial layer. The increase in diameter of the aorta was reversed to healthy controls. Moreover, PGG treatment restored degraded elastic lamina and increased the circumferential strain of aneurysmal aorta to the healthy levels. CONCLUSION: Our results support that site-specific delivery of PGG with targeted nanoparticles can be used to treat already developed AAA. Such therapy can reverse inflammatory markers and restore arterial homeostasis.

The lysyl oxidase like 2/3 enzymatic inhibitor, PXS-5153A, reduces crosslinks and ameliorates fibrosis.

Fibrosis is characterized by the excessive deposition of extracellular matrix and crosslinked proteins, in particular collagen and elastin, leading to tissue stiffening and disrupted organ function. Lysyl oxidases are key players during this process, as they initiate collagen crosslinking through the oxidation of the ε-amino group of lysine or hydroxylysine on collagen side-chains, which subsequently dimerize to form immature, or trimerize to form mature, collagen crosslinks. The role of LOXL2 in fibrosis and cancer is well documented, however the specific enzymatic function of LOXL2 and LOXL3 during disease is less clear. Herein, we describe the development of PXS-5153A, a novel mechanism based, fast-acting, dual LOXL2/LOXL3 inhibitor, which was used to interrogate the role of these enzymes in models of collagen crosslinking and fibrosis. PXS-5153A dose-dependently reduced LOXL2-mediated collagen oxidation and collagen crosslinking in vitro. In two liver fibrosis models, carbon tetrachloride or streptozotocin/high fat diet-induced, PXS-5153A reduced disease severity and improved liver function by diminishing collagen content and collagen crosslinks. In myocardial infarction, PXS-5153A improved cardiac output. Taken together these results demonstrate that, due to their crucial role in collagen crosslinking, inhibition of the enzymatic activities of LOXL2/LOXL3 represents an innovative therapeutic approach for the treatment of fibrosis.

Pentagalloyl glucose increases elastin deposition, decreases reactive oxygen species and matrix metalloproteinase activity in pulmonary fibroblasts under inflammatory conditions.

Emphysema is characterized by degradation of lung alveoli that leads to poor airflow in lungs. Irreversible elastic fiber degradation by matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) activity leads to loss of elasticity and drives the progression of this disease. We investigated if a polyphenol, pentagalloyl glucose (PGG) can increase elastin production in pulmonary fibroblasts. We also studied the effect of PGG treatment in reducing MMP activity and ROS levels in cells. We exposed rat pulmonary fibroblasts to two different types of inflammatory environments i.e., tumor necrosis factor-α (TNF-α) and cigarette smoke extract (CSE) to mimic the disease. Parameters like lysyl oxidase (LOX) and elastin gene expression, MMP-9 activity in the medium, lysyl oxidase (LOX) activity and ROS levels were studied to assess the effect of PGG on pulmonary fibroblasts. CSE inhibited lysyl oxidase (LOX) enzyme activity that resulted in a decreased elastin formation. Similarly, TNF-α treated cells showed less elastin in the cell layers. Both these agents caused increase in MMP activity and ROS levels in cells. However, when supplemented with PGG treatment along with these two inflammatory agents, we saw a significant increase in elastin deposition, reduction in both MMP activity and ROS levels. Thus PGG, which has anti-inflammatory, anti-oxidant properties coupled with its ability to aid in elastic fiber formation, can be a multifunctional drug to potentially arrest the progression of emphysema.

MicroRNA-181b Controls Atherosclerosis and Aneurysms Through Regulation of TIMP-3 and Elastin.

RATIONALE: Atherosclerosis and aneurysms are leading causes of mortality worldwide. MicroRNAs (miRs) are key determinants of gene and protein expression, and atypical miR expression has been associated with many cardiovascular diseases; although their contributory role to atherosclerotic plaque and abdominal aortic aneurysm stability are poorly understood. OBJECTIVE: To investigate whether miR-181b regulates tissue inhibitor of metalloproteinase-3 expression and affects atherosclerosis and aneurysms. METHODS AND RESULTS: Here, we demonstrate that miR-181b was overexpressed in symptomatic human atherosclerotic plaques and abdominal aortic aneurysms and correlated with decreased expression of predicted miR-181b targets, tissue inhibitor of metalloproteinase-3, and elastin. Using the well-characterized mouse atherosclerosis models of Apoe-/- and Ldlr-/-, we observed that in vivo administration of locked nucleic acid anti-miR-181b retarded both the development and the progression of atherosclerotic plaques. Systemic delivery of anti-miR-181b in angiotensin II-infused Apoe-/- and Ldlr-/- mice attenuated aneurysm formation and progression within the ascending, thoracic, and abdominal aorta. Moreover, miR-181b inhibition greatly increased elastin and collagen expression, promoting a fibrotic response and subsequent stabilization of existing plaques and aneurysms. We determined that miR-181b negatively regulates macrophage tissue inhibitor of metalloproteinase-3 expression and vascular smooth muscle cell elastin production, both important factors in maintaining atherosclerotic plaque and aneurysm stability. Validation studies in Timp3-/- mice confirmed that the beneficial effects afforded by miR-181b inhibition are largely tissue inhibitor of metalloproteinase-3 dependent, while also revealing an additional protective effect through elevating elastin synthesis. CONCLUSIONS: Our findings suggest that the management of miR-181b and its target genes provides therapeutic potential for limiting the progression of atherosclerosis and aneurysms and protecting them from rupture.

Elastin-Derived Peptides Promote Abdominal Aortic Aneurysm Formation by Modulating M1/M2 Macrophage Polarization.

Abdominal aortic aneurysm is a dynamic vascular disease characterized by inflammatory cell invasion and extracellular matrix degradation. Damage to elastin in the extracellular matrix results in release of elastin-derived peptides (EDPs), which are chemotactic for inflammatory cells such as monocytes. Their effect on macrophage polarization is less well known. Proinflammatory M1 macrophages initially are recruited to sites of injury, but, if their effects are prolonged, they can lead to chronic inflammation that prevents normal tissue repair. Conversely, anti-inflammatory M2 macrophages reduce inflammation and aid in wound healing. Thus, a proper M1/M2 ratio is vital for tissue homeostasis. Abdominal aortic aneurysm tissue reveals a high M1/M2 ratio in which proinflammatory cells and their associated markers dominate. In the current study, in vitro treatment of bone marrow-derived macrophages with EDPs induced M1 macrophage polarization. By using C57BL/6 mice, Ab-mediated neutralization of EDPs reduced aortic dilation, matrix metalloproteinase activity, and proinflammatory cytokine expression at early and late time points after aneurysm induction. Furthermore, direct manipulation of the M1/M2 balance altered aortic dilation. Injection of M2-polarized macrophages reduced aortic dilation after aneurysm induction. EDPs promoted a proinflammatory environment in aortic tissue by inducing M1 polarization, and neutralization of EDPs attenuated aortic dilation. The M1/M2 imbalance is vital to aneurysm formation.

4-Hydroxynonenal impairs transforming growth factor-ß1-induced elastin synthesis via epidermal growth factor receptor activation in human and murine fibroblasts.

Elastin is a long-lived protein and a key component of connective tissues. The tissular elastin content decreases during chronological aging, and the mechanisms underlying its slow repair are not known. Lipid oxidation products that accumulate in aged tissues may generate protein dysfunction. We hypothesized that 4-hydroxynonenal (4-HNE), a highly reactive α,ß-aldehydic product generated from polyunsaturated fatty acid peroxidation, could contribute to inhibiting elastin repair by antagonizing the elastogenic signaling of transforming growth factor-ß1 (TGF-ß1) in skin fibroblasts. We report that a low 4-HNE concentration (2µmol/L) inhibits the upregulation of tropoelastin expression stimulated by TGF-ß1 in human and murine fibroblasts. The study of signaling pathways potentially involved in the regulation of elastin expression showed that 4-HNE did not block the phosphorylation of Smad3, an early step of TGF-ß1 signaling, but inhibited the nuclear translocation of Smad2. Concomitantly, 4-HNE modified and stimulated the phosphorylation of the epidermal growth factor receptor (EGFR) and subsequently ERK1/2 activation, leading to the phosphorylation/stabilization of the Smad transcriptional corepressor TGIF, which antagonizes TGF-ß1 signaling. Inhibitors of EGFR (AG1478) and MEK/ERK (PD98059), and EGFR-specific siRNAs, reversed the inhibitory effect of 4-HNE on TGF-ß1-induced nuclear translocation of Smad2 and tropoelastin synthesis. In vivo studies on aortas from aged C57BL/6 mice showed that EGFR is modified by 4-HNE, in correlation with an increased 4-HNE-adduct accumulation and decreased elastin content. Altogether, these data suggest that 4-HNE inhibits the elastogenic activity of TGF-ß1, by modifying and activating the EGFR/ERK/TGIF pathway, which may contribute to altering elastin repair in chronological aging and oxidative stress-associated aging processes.

Self-assembly of elastin-based peptides into the ECM: the importance of integrins and the elastin binding protein in elastic fiber assembly.

The formation of a suitable extracellular matrix (ECM) that promotes cell adhesion, organization, and proliferation is essential within biomaterial scaffolds for tissue engineering applications. In this work, short elastin mimetic peptide sequences, EM-19 and EM-23, were engineered to mimic the active motifs of human elastin in hopes that they can stimulate ECM development in synthetic polymer scaffolds. Each peptide was incubated with human aortic smooth muscle cells (SMCs) and elastin and desmosine production were quantified after 48 h. EM-19 inhibited elastin production through competitive binding phenomena with the elastin binding protein (EBP), whereas EM-23, which contains an RGDS domain, induces recovery of elastin production at higher concentrations, alluding to a higher binding affinity for the integrins than for the EBP and the involvement of integrins in elastin production. Colocalization of each peptide with the elastin matrix was confirmed using immunofluorescent techniques. Our data suggest that with appropriate cell-binding motifs, we can simulate the cross-linking of tropoelastin into the developing elastin matrix using short peptide sequences. The potential for increased cell adhesion and the incorporation of elastin chains into tissue engineering scaffolds make these peptides attractive bioactive moieties that can easily be incorporated into synthetic biomaterials to induce ECM formation.

Modifications of arterial phenotype in response to amine oxidase inhibition by semicarbazide.

Semicarbazide-sensitive amine oxidase (SSAO)-deficient mice present no alteration in elastin cross-linking processes and carotid mechanical properties. In contrast, previous studies have shown that SSAO inhibitors induced marked anomalies in arterial structure and function. The aim of the present study was to examine the effect of semicarbazide (SCZ), an efficient SSAO inhibitor, on the arterial phenotype of the carotid artery in relation to modulation of SSAO and lysyl oxidase activities in growing rats. We first show that after 6 weeks of SCZ treatment (100 mg/kg per day), SSAO activity was reduced by 90%, whereas lysyl oxidase activity was only partially inhibited (<60%) in carotid artery, compared with controls. There was significant growth inhibition and no difference in mean arterial pressure but an increase in pulse pressure with a smaller arterial diameter in SCZ-treated rats. SCZ decreased aortic insoluble elastin without a change in total collagen. In addition, extracellular proteins other than insoluble elastin and collagen were increased in SCZ-treated rats. All of the elastic lamellae presented globular masses along their periphery, and focal disorganization was observed in the ascending aorta. Carotid artery mechanical strength was lower in SCZ-treated rats, and the elastic modulus-wall stress curve was shifted leftward compared with controls, indicating increased stiffness. Thus, SCZ modifies arterial geometry and mechanical properties, alters elastic fiber structure, and reduces the content of cross-linked elastin. Because these abnormalities are essentially absent in SSAO-deficient mice, our results suggest that lysyl oxidase inhibition is responsible for the major part of the vascular phenotype of SCZ-treated rats.

Basic fibroblast growth factor suppresses tropoelastin gene expression in cultured human periodontal fibroblasts.

Growth factors are known to play a major role in the regeneration of the periodontium. Basic fibroblast growth factor (bFGF) is a polypeptide growth factor considered to have a role in chemotaxis and mitogenesis of periodontal ligament (PDL) cells. The aim of this study was to assess the effect of bFGF on the transcription level of tropoelastin. As known controls, we assessed the transcription levels of collagen type I, collagen type II and the housekeeping gene, actin. Initially, PDL cells were cultured without bFGF for 3, 7 and 14 days. At each time point. total RNA was extracted and the levels of transcription were assessed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay. The results showed that tropoelastin mRNA is transcribed in PDL cells and its levels increased from minimal amounts by day 3 to maximal amounts by day 14 of culture. We further examined the effect of the addition of 10 ng/ml bFGF to the culture media by day 14. The results showed that the addition of bFGF suppressed the transcription level of tropoelastin. At that time, as expected, a decrease in collagen type I transcription level was shown, while the transcription level of collagen type III was not affected. The findings that elastin is transcribed in vitro by PDL cells, but only negligibly in vivo, imply mechanisms that downregulate or even shut down the expression of the elastin gene in the functioning PDL. Basic FGF might be one of the cytokines involved in control of elastin expression in vivo.

Inhibition by protease inhibitors of chemotaxis induced by elastin-derived peptides.

We studied the effect of two inhibitors of human neutrophil proteases on neutrophil chemotaxis induced by the hexapeptide Val-Gly-Val-Ala-Pro-Gly (VGVAPG), a recurring sequence in the elastin molecule. The inhibitors were tosyl-Phe chloromethyl ketone (TFCK) and N-methoxysuccinyl-Ala-Ala-Pro-Val chloromethyl ketone (MAAPVCK). We assayed chemotactic activity by the double-membrane technique in a modified Boyden chamber, after incubating the cells for 1 hr with varying concentrations of inhibitor. We observed a concentration-dependent inhibitory effect. We also measured the potency of the two chloromethyl ketones as protease inhibitors. The more potent protease inhibitor, MAAPVCK, was also the more effective in inhibiting VGVAPG-induced chemotaxis; its inhibitor dissociation constant was KI = 28 nM with elastase and KI = 33 nM with cathepsin G. For TFCK the corresponding KI values were 37 microM and 200 microM. The incubating concentration required to lower chemotaxis by half its uninhibited value was C0.5 = 0.64 microM for MAAPVCK, compared to C0.5 = 3.4 microM for TFCK. A third peptide, triglycinate (gly3), which did not inhibit the proteolytic activity of either elastase or cathepsin G, did not inhibit chemotaxis. Chemotaxis induced by formyl Met-Leu-Phe (fMLP) was weakly inhibited by both chloromethyl ketones with TFCK being somewhat more effective than MAAPVCK. We concluded that inhibition of VGVAPG-induced chemotaxis is in part specific, receptor mediated. We suggest that proteolytic inhibitors protect the extracellular matrix from degradation by inhibiting chemotaxis. Comparing the inhibitor concentrations required to half proteolytic activity with the concentration required to half chemotactic activity, we further suggest that the two functions may be of comparable significance.

Partial characterization of matrix-associated serine protease inhibitors from human skin cells.

Serine protease inhibitors have important regulatory roles in angiogenesis, intravascular fibrinolysis, wound healing, and cell migration. In this study, the extracellular matrix secreted by cultured human keratinocytes, foreskin fibroblasts, and SV-40-transformed human skin fibroblasts was analyzed for serine protease inhibitors by substrate reverse zymography. We found that the extracellular matrix deposited by these cells contained three inhibitors (M(r) 33,000, 31,000, and 27,000). These inhibitors protected the degradation of gelatin by trypsin and elastase, and of casein by plasmin. In contrast, the gelatinolytic activities of thermolysin and papain were not inhibited. Compared to untreated cells, cells treated with phorbol 12-myristate 13-acetate showed a two- to 10-fold increase in the expression of these inhibitors. Cycloheximide and actinomycin D decreased the cellular expression of these inhibitors, suggesting the involvement of de novo protein and mRNA synthesis. Antitrypsin activity of these inhibitors was resistant to heat and sodium dodecylsulfate, but was lost after reduction of disulfide bonds. The inhibitors bound specifically to trypsin and could be eluted from a trypsin column in active form. Collectively, these data suggest that the extracellular matrix deposited by keratinocytes and dermal fibroblasts contains active serine protease inhibitors.

[Inhibition of elastin hydrolysis, catalyzed by human leukocyte elastase and cathepsin G, by the Bowman-Birk type soy inhibitor]. / Ingibirovanie soevym ingibitorom tipa Baumana-Birk gidorliza élastina, kataliziruemogo élastazoi i katepsinom G iz leikotsitov cheloveka.

Cathepsin G stimulates the hydrolysis of elastin from bovine neck ligament catalyzed by human leukocyte elastase. Stimulation factor depends on the ratio of the enzyme concentrations and ionic strength and equals 1.0-2.0. The classical Bowman-Birk inhibitor from soya retards strongly the hydrolysis of elastin catalyzed by leukocyte elastase, cathepsin G and the mixture of both. The inhibitory effect is practically unaffected by the adsorption of the enzymes on elastin, prolongation of the enzymatic reaction and ionic strength.

Inhibition of elastin synthesis by high potassium salt is mediated by Ca2+ influx in cultured smooth muscle cells in vitro: reciprocal effects of K+ on elastin and collagen synthesis.

Elastin synthesis in cultured smooth muscle cells was inhibited by one fourth in the presence of 0.1 M K+ in the medium. The degree of inhibition paralleled the decrease in the steady-state levels of elastin mRNA. The inhibition of elastin synthesis was blocked by addition of 1 microM nifedipine, a Ca2+ antagonist. Comparable inhibition of elastin synthesis was observed by addition of A23187, a Ca2+ ionophore. In contrast, collagen synthesis and thymidine uptake were stimulated threefold and twofold respectively in the presence of 0.1 M K+ with a concomitant increase in collagen mRNA. The stimulation of collagen synthesis was also blocked by nifedipine. These results indicate that K+ modulates elastin and collagen synthesis and their gene expression reciprocally, and these effects are mediated by Ca2+ influx. Thus K+ exerts profound effects on the composition of extracellular matrices in aorta.

Enalapril suppresses normal accumulation of elastin and collagen in cardiovascular tissues of growing rats.

We have investigated the effect of enalapril, an angiotensin converting-enzyme (ACE) inhibitor, on the accumulation of ventricular and vascular collagen and elastin in young, growing rats. Beginning at either 4 or 10 wk of age, male Wistar rats were treated with enalapril for 2 or 5 wk. Enalapril treatment had no significant effect on body weight and small, generally non-significant effects on systolic and diastolic blood pressures. In contrast, young enalapril-treated animals showed a marked decrease in accumulation of total elastin and collagen in both large (aorta, renal, and carotid) and smaller (superior and large mesenteric) arteries, as well as a large reduction in total collagen in both left and right ventricles. This effect also was present but less pronounced in rats treated with enalapril beginning at 10 wk of age. These data indicate that inhibition of ACE activity during a period of rapid growth significantly reduces accumulation of vascular and ventricular connective tissue and suggests that angiotensin II may be important in normal cardiovascular development and growth.

Recombinant interleukin-1 beta inhibits elastin formation by a neonatal rat lung fibroblast subtype.

The effect of recombinant interleukin-1 beta (rIL-1 beta) on elastin accumulation by lipid-laden interstitial cells (LIC) derived from neonatal rat lung was examined. The LIC, a fibroblast subtype, synthesized large amounts of elastin which was deposited into the extracellular matrix. This elastin was alkali-resistant and had an amino acid composition typical of adult rat elastin. Treatment of lipid-laden interstitial cell cultures with rIL-1 beta at 100 pg/ml caused a dramatic decrease in elastin accumulation as assessed by hot alkali treatment and transmission electron micrographs of the cell cultures. Tropoelastin formation was selectively decreased by rIL-1 beta relative to other proteins. Steady state levels of elastin mRNA were slightly decreased by rIL-1 beta at 5 pg/ml and markedly decreased by rIL-1 beta at 50 pg/ml or greater. The addition of indomethacin had no effect on rIL-1 beta-induced decreases in elastin mRNA levels. Inhibiting protein synthesis with cycloheximide blocked the effect of rIL-1 beta on elastin mRNA levels. The level of alpha 1(I) collagen mRNA was decreased by rIL-1 beta, but only at concentrations higher than that needed to induce a decrease in elastin mRNA. These data indicate that rIL-1 beta decreased steady state levels for elastin mRNA and elastin accumulation and can selectively regulate the accumulation of elastin and collagen.

Cigarette smoke impairs elastin resynthesis in lungs of hamsters with elastase-induced emphysema.

The severity of pulmonary emphysema can be affected by exposure to cigarette smoke in several ways. Inactivation of alpha-1-antitrypsin and recruitment of leukocytes to lung airways shifts the protease-antiprotease balance towards increased elastolytic activity. The present study demonstrates an additional effect of cigarette smoke inhalation and retardation of the repair process and of the neosynthesis of cross-linked elastin. Hamsters with elastase-induced emphysema, exposed to cigarette smoke for 1 wk immediately after elastase administration, showed a 40% reduction of 14C-lysine incorporation into the elastin-specific cross-links, desmosine, and isodesmosine. Concomitantly, there was a decrease in the level of lung lysyl oxidase to that observed in uninjured control animals, in sharp contrast to the sevenfold increase in lysyl oxidase activity in hamsters with elastase-induced emphysema recovering under atmospheric conditions. These findings suggest that impairment of the production of lysyl oxidase and the resynthesis of cross-linked elastin by smoke inhalation exacerbates alveolar destruction.