Metallothionein (MT) plays a significant role in heavy metal removal, antioxidant defense, and immune regulation. The current predominant approach for obtaining natural MT is extraction from tissue, which often entails complex procedures resulting in limited yields. In recent years, researchers have adopted the strategy of fusing labels such as GST or His for the heterologous expression of MT. However, a challenge in industrial production arises from the subsequent removal of these labels, which often leads to a significant reduction in the yield. The fusion with elastin-like polypeptides (ELPs) offers a promising solution for achieving soluble expression of the target protein, while providing a simple and fast purification process. In this study, ELP was fused with MT, which significantly up-regulated the soluble expression of MT. The fusion protein ELP-MT with the purity above 97% was obtained efficiently and simply by inverse transition cycling (ITC). ELP-MT exhibited a remarkable 2,2'-azinobis(3-ethylbenzothiazoline-6- sulfonic acid) ammonium salt (ABTS) scavenging activity, with the half maximal inhibitory concentration (IC₅₀) of 0.77 μmol/L, which was 53.7 times that of the vitamin E derivative Trolox. In addition, the fusion protein demonstrated strong 1,1-diphenyl-2-trinitrohydrazine (DPPH) scavenging ability. Furthermore, ELP-MT had no toxicity to the proliferation and promoted the adhesion and migration of NIH/3T3 cells. All these results indicated that ELP-MT had good biocompatibility. We constructed the fusion protein ELP-MT combining the unique properties of MT and elastin, laying a technical foundation for the large-scale production of recombinant MT and facilitating the applications in food, health supplement, and cosmetic industries.
Metallothionein/metabolism* ; Elastin/chemistry* ; Recombinant Fusion Proteins/pharmacology* ; Mice ; Animals ; Peptides/metabolism* ; Escherichia coli/metabolism* ; NIH 3T3 Cells

OBJECTIVE: To identify whether naringenin plays a protective role during thoracic aneurysm formation in Marfan syndrome. METHODS: To validate the effect of naringenin, Fbn1^C1039G/+ mice, the mouse model of Marfan syndrome, were fed with naringenin, and the disease progress was evaluated. The molecular mechanism of naringenin was further investigated via in vitro studies, such as bioluminescence resonance energy transfer (BRET), atomic force microscope and radioligand receptor binding assay. RESULTS: Six-week-old Fbn1^C1039G/+ mice were fed with naringenin for 20 weeks. Compared with the control group, naringenin significantly suppressed the aortic expansion [Fbn1^C1039G/+ vs. Fbn1^C1039G/++naringenin: (2.49±0.47) mm, n=18 vs. (1.87±0.19) mm, n=22, P < 0.05], the degradation of elastin, and the expression and activity of matrix metalloproteinase 2 (MMP2) and MMP9 in the ascending aorta of Fbn1^C1039G/+ mice. Besides, treatment with naringenin for 6 weeks also attenuated the disease progress among the 20-week-old Fbn1^C1039G/+ mice with established thoracic aortic aneurysms [Fbn1^C1039G/+ vs. Fbn1^C1039G/++naringenin: (2.24±0.23) mm, n=8 vs. (1.90±0.17) mm, n=8, P < 0.05]. To understand the underlying molecular mechanisms, we examined the effects of naringenin on angiotensin Ⅱ type 1 receptor (AT1) signaling and transforming growth factor-β (TGF-β) signaling respectively, which were the dominant signaling pathways contributing to aortopathy in Marfan syndrome as previously reported. The results showed that naringenin decreased angiotensin Ⅱ (Ang Ⅱ)-induced phosphorylation of protein kinase C (PKC) and extracellular regulating kinase 1/2 (ERK1/2) in HEK293A cell overexpressing AT1 receptor. Moreover, naringenin inhibited Ang Ⅱ-induced calcium mobilization and uclear factor of activated T-cells (NFAT) signaling. The internalization of AT1 receptor and its binding to β-arrestin-2 with Ang Ⅱ induction were also suppressed by naringenin. As evidenced by atomic force microscope and radioligand receptor binding assay, naringenin inhibited Ang Ⅱ binding to AT1 receptor. In terms of TGF-β signaling, we found that feeding the mice with naringenin decreased the phosphorylation of Smad2 and ERK1/2 as well as the expression of TGF-β downstream genes. Besides, the serum level of TGF-β was also decreased by naringenin in the Fbn1^C1039G/+ mice. Furthermore, we detected the effect of naringenin on platelet, a rich source of TGF-β, both in vivo and in vitro. And we found that naringenin markedly decreased the TGF-β level by inhibiting the activation of platelet. CONCLUSION Our study showed that naringenin has a protective effect on thoracic aortic aneurysm formation in Marfan syndrome by suppressing both AT1 and TGF-β signaling.
Angiotensin II/metabolism* ; Animals ; Aortic Aneurysm, Thoracic/prevention & control* ; Calcium/metabolism* ; Disease Models, Animal ; Elastin/metabolism* ; Fibrillin-1/metabolism* ; Flavanones ; Marfan Syndrome/metabolism* ; Matrix Metalloproteinase 2 ; Matrix Metalloproteinase 9 ; Mice ; Mice, Inbred C57BL ; Protein Kinase C/metabolism* ; Receptor, Angiotensin, Type 1/metabolism* ; Transforming Growth Factor beta/metabolism* ; Transforming Growth Factors/metabolism* ; beta-Arrestins/metabolism*

BACKGROUNDCoronary artery damage from Kawasaki disease (KD) is closely linked to the dysfunction of the endothelial progenitor cells (EPCs). The aim of the present study was to evaluate the modulatory effect of granulocyte colony-stimulating factor (G-CSF) on EPCs and elastin breakdown of coronary arteries in a KD mouse model.

METHODSA Lactobacillus casei cell wall extract (LCWE)-induced KD model was established in C57BL/6 mice that were subsequently administrated with recombinant human G-CSF (rhG-CSF). Nω-nitro-L-arginine methyl ester (L-NAME) was administrated for the negative intervention. Evaluations included coronary artery lesions, EPC number and functions, and the plasma concentration of nitric oxide (NO).

RESULTSElastin breakdown was found in the coronary arteries of model mice 56 days after injection of LCWE. The number of circulating EPCs, plasma concentration of NO, and functions of bone marrow EPCs, including proliferation, adhesion, and migration abilities, were all lower in the KD model group compared with those in the control group. After administration of rhG-CSF, the number of circulating EPCs and plasma concentration of NO were increased significantly compared with those in the KD model group. There were also increases in the functional indexes of EPCs. Furthermore, rhG-CSF administration improved the elastin breakdown effectively. However, these protective effects of rhG-CSF on coronary arteries were attenuated by L-NAME.

CONCLUSIONThe present study indicated that the administration of G-CSF prevents elastin breakdown of the coronary arteries by enhancing the number and functions of EPCs via the NO system, and then accelerates the repair of coronary artery lesions in the KD.

Animals ; Coronary Vessels ; cytology ; drug effects ; metabolism ; Disease Models, Animal ; Elastin ; metabolism ; Endothelial Progenitor Cells ; cytology ; Granulocyte Colony-Stimulating Factor ; therapeutic use ; Male ; Mice ; Mice, Inbred C57BL ; Mucocutaneous Lymph Node Syndrome ; blood ; drug therapy ; metabolism ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitrogen Oxides ; blood

BACKGROUNDThe pathological characteristics of abdominal aortic aneurysm (AAA) involved the regression of extracellular matrix (ECM) in aortic walls, especially elastic structure in medial layer. As the major structural protein of aorta, elastin contributes to the extensibility and elastic recoil of the vessels. We hypothesized that overexpression of elastin in vessel walls might regenerate the elastic structure of ECM, restore the elastic structure of the aneurysmal wall, and eventually lead to a reduction of aortic diameters (ADs) in an experimental model of AAA.

METHODSTropoelastin (TE) of Sprague Dawley (SD) rat was synthesized by reverse transcription polymerase chain reaction and used to construct adneviral vectors containing elastin precursor protein (AdTE-GFP). Cultured vascular smooth muscle cells (VSMCs) from aortas of male SD rats were transfected with AdTE-GFP, AdGFP, adenoviral vector (AdNull), and phosphate buffered saline (PBS). Immunofluorescence staining was performed to determine the expression of elastin in transfected cells. The expression of elastic fibers in ECM of VSMCs transfected with AdTE-GFP were detected by fluorescence microscopy and transmission electron microscopy (TEM) at 1, 3, and 5 days following gene transfer. The AAA vessel walls were infused with AdTE-GFP or an empty AdNull, or PBS directly into the aneurysmal lumen. ADs of the aneurysms were compared in infused aortas. Formation of new elastic fibers in vivo was assessed by hematoxylin and eosin, and elastic von-Giesson staining. Recombinant elastin-GFP in vivo was identified by immunohistochemical staining.

RESULTSElastic fibers were increased both in ECM of VSMC and in vessel walls after gene transfer. Histological studies revealed that the AdTE-GFP-transduced aortas had elastic fiber regeneration in the aneurysmal walls. The AdTE-GFP-transduced aortas showed a decreased AD (23.04% ± 14.49%, P < 0.01) in AAA vessel walls.

CONCLUSIONSElastic fibers have been successfully overexpressed both in vitro and in a rat model of AAA by a technique of gene transfer. The overexpression of elastic fibers within the aneurysmal tissue appeared to reverse the aneurysm dilatation in this model.

Animals ; Aortic Aneurysm, Abdominal ; metabolism ; therapy ; Elastic Tissue ; metabolism ; Elastin ; genetics ; metabolism ; Fluorescent Antibody Technique ; Male ; Muscle, Smooth, Vascular ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tropoelastin ; genetics ; metabolism

BACKGROUNDElastin derived peptides can regulate melanocyte precursor development. Ultraviolet irradiation, infrared radiation and heat can increase the synthesis of tropoelastin in human skin epidermis. The aim of this study was to investigate whether the over expressed tropoelastin in epidermis has some role in melanogenesis of melanocytes.

METHODSA375 human melanoma cells were treated with different concentrations of kappa elastin for 24 hours. A375 human melanoma cells were randomly assigned to control, kappa elastin, and lactose pre-incubated groups. The cell viabilities were detected by the methyl thiazoleterazolium assay. Melanin content and tyrosinase activity in A375 melanoma cells were measured. The expressions of endothelin B receptor (ET(B)R) mRNA and c-kit mRNA in A375 melanoma cells were measured by quantative reverse transcription polymerase chain reaction.

RESULTSFifty µg/ml of kappa elastin significantly increased the melanin content by 56.64% compared with the control (P < 0.05). Kappa elastin increased cellular tyrosinase activity by 46.73% compared with the control at 24 hours (P < 0.05). Kappa elastin increased the expressions of ET(B)R and c-kit mRNA levels by 2.13-fold and 2.47-fold compared with the controls, respectively. When pre-incubating cells with a lactose solution (10 mmol/L), the inhibition on melanin production was 34.96% compared with the kappa elastin group (P < 0.05), tyrosinase activity was inhibited by 29.93% compared with kappa elastin group (P < 0.05), and the expressions of ET(B)R mRNA and c-kit mRNA were decreased by 1.56-fold and 0.82-fold compared with kappa elastin group, respectively.

CONCLUSIONKappa elastin increased the melanogenesis in A375 melanoma cells via the stimulation of tyrosinase activity and the expression of ET(B)R and c-kit. The over expressed tropoelastin produced by keratinocytes might play a role in melanogenesis of epidermal melanocytes.

Cell Line, Tumor ; Cell Survival ; drug effects ; Elastin ; pharmacology ; Humans ; Keratinocytes ; drug effects ; Melanins ; metabolism ; Melanoma ; Proto-Oncogene Proteins c-kit ; metabolism ; Receptor, Endothelin B ; metabolism

The elastin metabolism in systemic sclerosis (SSc) has been known to be abnormal. The authors investigated relationship between the clinical manifestations of systemic sclerosis (SSc) and serum levels of soluble elastin-derived peptide (S-EDP) and anti-elastin antibodies. Serum samples were obtained from 79 patients with SSc and 79 age- and sex-matched healthy controls. Concentrations of serum S-EDP and anti-elastin antibodies were measured by ELISA. The serum concentrations of S-EDP in SSc patients were significantly higher than in healthy controls (median, 144.44 ng/mL vs 79.59 ng/mL, P < 0.001). Serum EDP concentrations were found to be correlated with disease duration in SSc (P = 0.002) and particularly in diffuse cutaneous SSc (P = 0.005). Levels of anti-elastin antibodies were found to be more elevated in SSc patients than in healthy controls (median, 0.222 U vs 0.191 U, P = 0.049), more increased in diffuse cutaneous SSc than limited cutaneous SSc (median, 0.368 U vs 0.204 U, P = 0.031). In addition, levels of anti-elastin antibodies were also found to be negatively associated with presence of anti-centromere antibody (P = 0.023). The S-EDP levels were not found to be correlated with levels of anti-elastin antibodies. The increased S-EDP and anti-elastin antibody levels and association with clinical and laboratory characteristics may reflect the abnormal metabolism in SSc.
Adult ; Antibodies, Anti-Idiotypic/*blood/immunology ; Centromere/immunology ; Elastin/*blood/immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Male ; Middle Aged ; Peptides/*blood/immunology ; Scleroderma, Systemic/*metabolism/pathology

BACKGROUNDCoronary artery damage from Kawasaki disease (KD) is closely linked to the dysfunction of endothelial progenitor cells (EPCs). The aim of the present study was to evaluate the therapeutic effect of EPCs transplantation in KD model.

METHODSLactobacillus casei cell wall extract (LCWE)-induced KD model in C57BL/6 mice was established. The model mice were injected intravenously with bone marrow-derived in vitro expanded EPCs. Histological evaluation, number of circulating EPCs and the function of bone marrow EPCs were examined at day 56.

RESULTSInflammation was found around the coronary artery of the model mice after 14 days, Elastin breakdown was observed after 56 days. CM-Dil labeled EPCs incorporated into vessel repairing foci was found. At day 56, the number of peripheral EPCs in the KD model group was lower than in EPCs transplanted and control group. The functional index of bone marrow EPCs from the KD model group decreased in proliferation, adhesion and migration. Increased number of circulating EPCs and improved function were observed on the EPCs transplanted group compared with model group.

CONCLUSIONExogenously administered EPCs, which represent a novel strategy could prevent the dysfunction of EPCs, accelerate the repair of coronary artery endothelium lesion and decrease the occurrence of aneurysm.

Animals ; Cell Adhesion ; physiology ; Cell Proliferation ; Disease Models, Animal ; Elastin ; metabolism ; Endothelial Cells ; cytology ; Male ; Mice ; Mucocutaneous Lymph Node Syndrome ; metabolism ; therapy ; Stem Cell Transplantation ; psychology ; Stem Cells ; cytology ; physiology

Elastin-like polypeptides (ELPs) are temperature sensitive biopolymers composed of a Val-Pro-Gly-Xaa-Gly pentapeptide repeat that derived from a structural motif found in mammalian elastin. It was a promising tag for recombinant protein purification. Here, we de novo designed a novel ELPs gene and cloned it into the modified expression vector pET-22b(+). Then, we transformed the recombinant expression vector pET-22b-ELPs into Escherichia coli BL21(DE3). Upon induction by Isopropyl beta-D-Thiogalactoside (IPTG), ELPs was expressed and purified by a non-chromatographic purification method named inverse temperature cycling. The influences of salts types and concentrations on ELPs were also determined. The results showed that the transition temperature of the [KV8F-20] decreased to 19 degrees C by 0.4 mmol/L Na2CO3. Due to its small molecular weight and sensitivity to salt, the ELPs might be a useful purification tag, which can provide a reliable and simple non-chromatographic method for purification of the recombinant protein by inverse transition cycling.
Chromatography ; Elastin ; biosynthesis ; genetics ; isolation & purification ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Peptides ; genetics ; isolation & purification ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; Sodium Chloride ; pharmacology

BACKGROUND: One of the characteristics of spinal stenosis is elastin degradation and fibrosis of the extracellular matrix of the ligamentum flavum. However, there have been no investigations to determine which biochemical factors cause these histologic changes. So we performed the current study to investigate the hypothesis that matrix metalloproteinases (MMPs), which possess the ability to cause extracellular matrix remodeling, may play a role as a mediator for this malady in the ligamentum flavum. METHODS: The ligamentum flavum specimens were surgically obtained from thirty patients with spinal stenosis, as well as from 30 control patients with a disc herniation. The extents of ligamentum flavum elastin degradation and fibrosis were graded (grade 0-4) with performing hematoxylin-eosin staining and Masson's trichrome staining, respectively. The localization of MMP-2 (gelatinase), MMP-3 (stromelysin) and MMP-13 (collagenase) within the ligamentum flavum tissue was determined by immunohistochemistry. The expressions of the active forms of MMP-2, MMP-3 and MMP-13 were determined by western blot analysis, and the blots were quantified using an imaging densitometer. The histologic and biochemical results were compared between the two conditions. RESULTS: Elastin degradation and fibrosis of the ligamentum flavum were significantly more severe in the spinal stenosis samples than that in the disc herniation samples (3.14 +/- 0.50 vs. 0.55 +/- 0.60, p < 0.001; 3.10 +/- 0.57 vs. 0.76 +/- 0.52, p < 0.001, respectively). The expressions of the active form of MMPs were identified in all the ligamentum flavums of the spinal stenosis and disc herniation patients. The expressions of active MMP-2 and MMP-13 were significantly higher in the spinal stenosis samples than that in the disc herniation samples (both p < 0.05). The expression of active MMP-3 was slightly higher in the spinal stenosis samples than that in the disc herniation samples, but the difference was not statistically significant (p = 0.131). MMP-2, -3, and -13 were positively stained on the ligamentum flavum fibroblasts. CONCLUSIONS: The current results suggest that the increased expression of active MMPs by the ligamentum flavum fibroblasts might be related to the elastin degradation and fibrosis of the ligamentum flavum in the patients who suffer with lumbar spinal stenosis.
Aged ; Blotting, Western ; Elastin/*metabolism ; Extracellular Matrix/metabolism/pathology ; Female ; Fibrosis ; Humans ; Immunohistochemistry ; Ligamentum Flavum/*metabolism/pathology ; *Lumbar Vertebrae ; Male ; Matrix Metalloproteinase 13/metabolism ; Matrix Metalloproteinase 2/metabolism ; Matrix Metalloproteinase 3/metabolism ; Matrix Metalloproteinases/*metabolism ; Middle Aged ; Spinal Stenosis/*metabolism/pathology

To understand the mechanical properties of aortic artery of atheroselerosis (AS), the aortic artery of rat with AS was studied by mechanical test. Wistar rats were used for establishing the model. The mechanical measurements of opening angle in zero-stress state and the vessel loading test were conducted on the isolated aortic arteries of AS rats. Data on the stress-strain of aortic artery were obtained. Determination of percentage of collagen content was made with the use of electron microscope. The relationship between mechanical measurements and collagen concentration was evaluated. The opening angle in the group of AS was significantly smaller than that in control (87.74 degrees +/-9.67 degrees vs. 196.03 degrees +/- 27.76 degrees, P < 0.001). Significant decrease of material constants (alpha0, alpha1, alpha2, b0, b1, b2) in both long axis and radial axis was observed in AS group(campared with control, P < 0.05-0.001). Close relationship between the mechanical constants and the percentage of elastin and collagen content was observed (r = -0.7523 to -0.8423, P < 0.001). In conclusion, mechanical remodeling in aortic artery of AS might be related with histological remodeling.
Animals ; Aorta, Abdominal ; metabolism ; pathology ; Atherosclerosis ; metabolism ; pathology ; physiopathology ; Biomechanical Phenomena ; Collagen ; analysis ; Elastin ; analysis ; Female ; Male ; Random Allocation ; Rats ; Rats, Wistar ; Stress, Mechanical