Degradation Of Extracellular Matrix Components Research Articles

A Microphysiological Model to Mimic the Placental Remodeling during Early Stage of Pregnancy under Hypoxia-Induced Trophoblast Invasion

Placental trophoblast invasion is critical for establishing the maternal–fetal interface, yet the mechanisms driving trophoblast-induced maternal arterial remodeling remain elusive. To address this gap, we developed a three-dimensional microfluidic placenta-on-chip model that mimics early pregnancy placentation in a hypoxic environment. By studying human umbilical vein endothelial cells (HUVECs) under oxygen-deprived conditions upon trophoblast invasion, we observed significant HUVEC artery remodeling, suggesting the critical role of hypoxia in placentation. In particular, we found that trophoblasts secrete matrix metalloproteinase (MMP) proteins under hypoxic conditions, which contribute to arterial remodeling by the degradation of extracellular matrix components. This MMP-mediated remodeling is critical for facilitating trophoblast invasion and proper establishment of the maternal–fetal interface. In addition, our platform allows real-time monitoring of HUVEC vessel contraction during trophoblast interaction, providing valuable insights into the dynamic interplay between trophoblasts and maternal vasculature. Collectively, our findings highlight the importance of MMP-mediated arterial remodeling in placental development and underscore the potential of our platform to study pregnancy-related complications and evaluate therapeutic interventions.

Investigation of multinucleation and apoptosis of macrophages of BCG-infected mice and their production of cathepsins and matrix metalloproteinases

The relevance of the study of the role of macrophages and their multinucleated forms in the pathogenesis of tuberculous granulomatosis is determined by its wide prevalence, the presence of severe socio-economic consequences of its morbidity and necrotic complications, which are based on the high destructive potential of macrophages associated with the role of hydrolases in the degradation of extracellular matrix components. Aim of the study was to investigate the features of the multinucleation, apoptosis and expression of a number of hydrolases in macrophages of BCG-infected mice. Material and methods. The intensity of macrophage multinucleation and apoptosis, the peculiarities of their expression of matrix metalloproteinases (MMP-1, MMP-9), catepsins (CatB, CatD), caspase-3, and p53 protein were studied in peritoneal cells cultures of intact and BCG-infected BALB/c mice. Results. The number of multinucleated macrophages increased according to the terms of the experiment, having a maximum value for 3 months of observation, but after 2 months almost reaching this level. The realization of apoptosis, multinucleation of macrophages had a complex character, determining the composition of their subpopulations. The dynamics of the expression of the studied hydrolases by macrophages indicated their unequal role in tissue necrosis at various stages of granulomogenesis. The high functional ability of multinucleated macrophages to produce hydrolases of certain types is shown. Intense expression of MMP-1 in the early stages of granulomogenesis and its maximum value, as well as CatD expression for 3 months, and strong expression of MMP-9 for 6 months were noted. Conclusions. Stimulation of plastic processes in macrophages under conditions of BCG-granulomatosis determines the formation of multinucleated macrophages with high functional potential and intensive expression of hydrolases by macrophages for 2 and 3 months of granulomogenesis. These are periods of high risk of necrotic complications of tuberculous granulomatosis, which should be taken into account when developing methods for their prevention and therapeutic correction.

Unravelling the role of cathepsins in cardiovascular diseases.

Lysosomal cathepsins as a regulatory medium have been assessed as potential therapeutic targets for the treatment of various cardiac diseases such as abdominal aortic aneurysm, hypertension, cardiomyopathy, coronary heart disease, atherosclerosis, etc. They are ubiquitous lysosomal proteases with papain-like folded protein structures that are involved in a variety of physiological processes, such as the digestion of proteins, activation of pro-inflammatory molecules, degradation of extracellular matrix components, and maturation of peptide hormones. Cathepsins are classified into three major groups: cysteine cathepsins, aspartic cathepsins, and serine-threonine cathepsins. Each of these groups is further divided into subgroups based on their substrate specificity, structural characteristics, and biochemical properties. Several studies suggest that cathepsins control the degradation of ECM components such as collagen and elastin fibres. These enzymes are highly expressed in macrophages and inflammatory cells, and their upregulation has been demonstrated to be critical in the progression of atherosclerotic lesions. Additionally, increased cathepsin activity has been linked to increased vascular inflammation and oxidative stress, both of which are associated with CVDs. Specifically, the inhibition of cathepsins may reduce the release of pro-apoptotic mediators such as caspase-3 and PARP-1, which are thought to contribute to plaque instability. The potential of cathepsins as biomarkers and therapeutic targets has also been supported by the identification of potential cathepsin inhibitors, which could be used to modulate the activities of cathepsins in a range of diseases. This review shall familiarise the readers with the role of cysteinyl cathepsins and their inhibitors in the pathogenesis of cardiovascular diseases.

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.

The disordered extracellular matrix landscape induced endometrial fibrosis of sheep: A multi-omics integrative analysis

Endometrial fibrosis leads to the destruction of endometrial function and affects reproductive performance. However, mechanisms underlying the development of endometrial fibrosis in sheep remain unclear. We use transcriptomic, proteomic, and metabolomic studies to reveal the formation mechanisms of endometrial fibrosis. The results showed that the fibrotic endometrial tissue phenotype presented fewer glands, accompanied by collagen deposition. Transcriptomic results indicated alterations in genes associated with the synthesis and degradation of extracellular matrix components, which alter metabolite homeostasis, especially in glycerophospholipid metabolism. Moreover, differentially expressed metabolites may play regulatory roles in key metabolic processes during fibrogenesis, including protein digestion and absorption, and amino acid synthesis. Affected by the aberrant genes, protein levels related to the extracellular matrix components were altered. In addition, based on Kyoto Encyclopedia of Genes and Genomes analysis of differentially expressed genes, metabolites and proteins, amino acid biosynthesis, glutathione, glycerophospholipid, arginine and proline metabolism, and cell adhesion are closely associated with fibrogenesis. Finally, we analyzed the dynamic changes in serum differential metabolites at different time points during fibrosis. Taken together, fibrosis development is related to metabolic obstacles in extracellular matrix synthesis and degradation triggered by disturbed gene and protein levels.

Matrix metalloproteinases system profile analysis and their endogenous inhibitors in children with periodontal diseases and various dysplasia phenotypes

Relevance. The search and development of modern non-invasive methods for early periodontal disease detection are of scientific and practical value for a personalized approach to disease course prognosis and explaining the choice of treatment tactics. Morphological immaturity, high intensity of anabolic processes, functional failure of neurohumoral, endocrine and immunological defence systems combined with an imbalance between the synthesis and degradation of extracellular matrix components in children with undifferentiated connective tissue disease (UCTD) significantly increases the risk of developing periodontal diseases. The available data on the activity of salivary matrix metalloproteinases (MMPs), which play a leading role in regulating connective tissue protein metabolism and bone remodelling mechanisms in children with UCTD of various degrees, are scarce and require further study.Purpose. The study aimed to explain the clinical, diagnostic and prognostic value of MMPs as markers of inflammatory periodontal diseases in children with UC TD and dysplastic disorders of various se verity.Material and methods. The study included 67 children with UCTD (main group) and 34 children of health groups I and II (comparison group) aged 12-17. The UCTD severity was established according to the diagnostic criteria by L. N. Abbakumova (2006) and a scale for assessing the significance of phenotypic and visceral signs. The main group was divided into two subgroups: Subgroup 1 (n = 38) – children with mild and moderate UCTD; Subgroup 2 (n=29) – children with severe UCTD. The studied groups had their periodontal status assessed, as well as the concentration of MMP-1, MMP-2, MMP-8, MMP-9 and their tissue inhibitors (TIMP-1, TIMP-2) in the oral fluid identified using the enzyme immunoassay, along with calculation done for the MMP ⁄ TIMP coefficients that set a balance between the degradation and synthesis of collagen.Results. The children with phenotypic signs of UCTD in the oral fluid appeared to show a statistically significant increase in the MMP-1, MMP-2, MMP-8, and MMP-9 levels and an imbalanced ratio in MMP-1⁄TIMP-1, MMP- 2⁄TIMP-2, MMP-8⁄TIMP-1, MMP-9⁄TIMP-1, compared with health groups I, II. An increase in the MMP-1, MMP-2, and MMP-8 concentrations, along with dominating expression of MMP-9 over TIMP-1 in the oral fluid of children of Subgroup 2, causes an increase in intensity and prevalence of periodontal diseases compared to the patients in Sub-group 1 and comparison group.Conclusion. Thus, when children with UCTD don’t demonstrate an increase in specific tissue inhibitors TIMP-1 and TIMP-2 in their oral fluid, along with an increase in the levels of the respective matrixins is the key pathogenetic factor in the degradation of extracellular matrix proteins, which causes a depressing impact on the proliferative activity in the periodontal set of tissues.

#111 : MMP-9 to TIMP-1 Expression Ratio on Endometrioma Favors Tissue Invasion: A Study on Chicken Chorioallantoic Membrane (CAM)

Background and Aims: Endometrioma occurs through the implantation of endometrial tissue in the ovary. The establishment of endometriotic lesions requires endometrial cells to invade the ovary by remodelling the extracellular matrix (ECM). It is plausible that these cells express protease. MMP-9 is a significant protease controlled by its natural inhibitor, TIMP-1. The balance between MMP-9 and TMIP-1 plays a critical role in the invasion of endometrioma that has never been investigated before. This study aimed to examine the correlation between MMP-9/TIMP-1 expression ratio and MMP-9 activity with the invasion of endometrioma tissue. Method: This study was conducted on 35 samples of endometrioma tissue obtained from 27 women with endometriosis at Dr Sardjito General Hospital, Yogjakarta. The tissue was then implanted into CAM to examine its invasion capability. Invasion scores were assessed using hematoxylin-eosin staining. MMP-9/TIMP-1 expressions ratio was analysed using western blot, while MMP-9 activity in degrading ECM components was determined using gelatin zymography. The Spearman correlation assesses the correlation between variables with significance set as p[Formula: see text]0.05. Results: The results showed a positive linear correlation between the MMP-9/TIMP-1 expression ratio and invasion scores of endometrioma tissue (r=0.486; p=0.003). In addition, the Kruskal-Wallis test showed there was a significant difference between the MMP-9/TIMP-1 expression ratio in each invasion score of endometrioma (p=0.032), and further analysis by Mann-Whitney post hoc tests revealed that significant differences were between invasion score 1 and 3 (p=0.037), also between invasion score 1 and 4 (p=0.010). MMP-9 activity had a strong positive linear correlation with invasion scores of endometrioma tissue (r=0.646; p=0.000). Conclusion: MMP-9 activity in degrading extracellular matrix promotes the invasion of endometrioma. The imbalance between MMP-9 and TIMP-1 enhances the capacity of endometrioma to break down ECM. Higher MMP-9 expression over TIMP-1 expression gives rise to the invasion of endometrioma.

Hepsin promotes breast tumor growth signaling via the TGFβ-EGFR axis.

Hepsin, a type II transmembrane serine protease, is commonly overexpressed in prostate and breast cancer. The hepsin protein is stabilized by the Ras-MAPK pathway, and, downstream, this protease regulates the degradation of extracellular matrix components and activates growth factor pathways, such as the hepatocyte growth factor (HGF) and transforming growth factor beta (TGFβ) pathway. However, how exactly active hepsin promotes cell proliferation machinery to sustain tumor growth is not fully understood. Here, we show that genetic deletion of the gene encoding hepsin (Hpn) in a WAP-Myc model of aggressive MYC-driven breast cancer inhibits tumor growth in the primary syngrafted sites and the growth of disseminated tumors in the lungs. The suppression of tumor growth upon loss of hepsin was accompanied by downregulation of TGFβ and EGFR signaling together with a reduction in epidermal growth factor receptor (EGFR) protein levels. We further demonstrate in 3D cultures of patient-derived breast cancer explants that both basal TGFβ signaling and EGFR protein expression are inhibited by neutralizing antibodies or small-molecule inhibitors of hepsin. The study demonstrates a role for hepsin as a regulator of cell proliferation and tumor growth through TGFβ and EGFR pathways, warranting consideration of hepsin as a potential indirect upstream target for therapeutic inhibition of TGFβ and EGFR pathways in cancer.

The novel HS-mimetic, Tet-29, regulates immune cell trafficking across barriers of the CNS during inflammation

BackgroundDisruption of the extracellular matrix at the blood–brain barrier (BBB) underpins neuroinflammation in multiple sclerosis (MS). The degradation of extracellular matrix components, such as heparan sulfate (HS) proteoglycans, can be prevented by treatment with HS-mimetics through their ability to inhibit the enzyme heparanase. The heparanase-inhibiting ability of our small dendrimer HS-mimetics has been investigated in various cancers but their efficacy in neuroinflammatory models has not been evaluated. This study investigates the use of a novel HS-mimetic, Tet-29, in an animal model of MS.MethodsNeuroinflammation was induced in mice by experimental autoimmune encephalomyelitis, a murine model of MS. In addition, the BBB and choroid plexus were modelled in vitro using transmigration assays, and migration of immune cells in vivo and in vitro was quantified by flow cytometry.ResultsWe found that Tet-29 significantly reduced lymphocyte accumulation in the central nervous system which, in turn, decreased disease severity in experimental autoimmune encephalomyelitis. The disease-modifying effect of Tet-29 was associated with a rescue of BBB integrity, as well as inhibition of activated lymphocyte migration across the BBB and choroid plexus in transwell models. In contrast, Tet-29 did not significantly impair in vivo or in vitro steady state-trafficking under homeostatic conditions.ConclusionsTogether these results suggest that Tet-29 modulates, rather than abolishes, trafficking across central nervous system barriers.

TGF- β Study on the Role of Iron Death Signal Transduction in Diabetes Nephropathy

DN (Diabetes Nephropathy) is the result of multiple factors, and its pathogenesis is complex. Extensive evidence has proven TGF- β. It plays an important role in the pathogenesis of DN, but the specific way of action is still unclear. TGF- β after binding and activating its receptor, its signal transmission needs to be carried out by a series of post receptor signal molecules TGF- β. It is the last and common important mediator of kidney damage caused by changes in biochemical factors and cytokines, such as blood glucose rise in diabetes. It can directly lead to DN characteristic pathological changes such as renal cell hypertrophy, excessive accumulation of extracellular matrix, glomerulosclerosis, renal interstitial fibrosis, etc. In DN, TGF- β the characteristic function of the β - lactamase is to stimulate the matrix deposition in the mesangial region. Through TGF- β. Further study on the role of DN iron death signal transduction in diabetes islets β the research on the mechanism of cell injury is reviewed. At the same time, it can not only increase the synthesis of extracellular matrix, but also inhibit the degradation of extracellular matrix components.

612 Proteomic and secretomic comparison of young and aged fibroblasts highlights cytoskeleton as a key component during aging

Crucial for skin homeostasis, synthesis and degradation of extracellular matrix components are orchestrated by dermal fibroblasts. During aging, alterations of component expression, such as collagens and enzymes, lead to reduction of the mechanical tension and defects of skin wound healing. The aim of this study was to better understand the molecular alterations underwent by fibroblasts during aging by comparing secretomic and proteomic signatures of fibroblasts from young (<35 years) and old (>55 years) skin donors, using LC-MS/MS.

272 (PB052) - Hepsin promotes breast tumor growth signaling via TGF beta-EGFR axis

Background: Hepsin, a type II transmembrane serine protease encoded by HPN gene, is commonly overexpressed in prostate and breast cancer. Hepsin protein is stabilized by Ras-MAPK pathway signaling, and downstream, this protease regulates the degradation of extracellular matrix components and activates growth factor pathways, including hepatocyte growth factor and transforming growth factor (TGF) beta pathway. The significance of the hepsin-dependent signaling on cell proliferation and tumor growth is still partially unclear.

The Role of Matrix Metalloproteinase in Inflammation with a Focus on Infectious Diseases.

Matrix metalloproteinases (MMPs) are involved in extracellular matrix remodeling through the degradation of extracellular matrix components and are also involved in the inflammatory response by regulating the pro-inflammatory cytokines TNF-α and IL-1β. Dysregulation in the inflammatory response and changes in the extracellular matrix by MMPs are related to the development of various diseases including lung and cardiovascular diseases. Therefore, numerous studies have been conducted to understand the role of MMPs in disease pathogenesis. MMPs are involved in the pathogenesis of infectious diseases through a dysregulation of the activity and expression of MMPs. In this review, we discuss the role of MMPs in infectious diseases and inflammatory responses. Furthermore, we present the potential of MMPs as therapeutic targets in infectious diseases.

Tumor extracellular matrix modulating strategies for enhanced antitumor therapy of nanomedicines

Nanomedicines have shown a promising strategy for cancer therapy because of their higher safety and efficiency relative to small-molecule drugs, while the dense extracellular matrix (ECM) in tumors often acts as a physical barrier to hamper the accumulation and diffusion of nanoparticles, thus compromising the anticancer efficacy. To address this issue, two major strategies including degrading ECM components and inhibiting ECM formation have been adopted to enhance the therapeutic efficacies of nanomedicines. In this review, we summarize the recent progresses of tumor ECM modulating strategies for enhanced antitumor therapy of nanomedicines. Through degrading ECM components or inhibiting ECM formation, the accumulation and diffusion of nanoparticles in tumors can be facilitated, leading to enhanced efficacies of chemotherapy and phototherapy. Moreover, the ECM degradation can improve the infiltration of immune cells into tumor tissues, thus achieving strong immune response to reject tumors. The adoptions of these two ECM modulating strategies to improve the efficacies of chemotherapy, phototherapy, and immunotherapy are discussed in detail. A conclusion, current challenges and outlook are then given.

Painful Temporomandibular Joint Clicking: Genetic Point of View.

To determine whether there is an association between gene polymorphisms and patients with painful temporomandibular joint (TMJ) clicking when compared to patients with painless TMJ clicking and a healthy control group. In this pilot study, the genotypic and allelic frequencies of candidate single-nucleotide polymorphisms (SNP) were compared among 60 individuals divided equally into three groups: patients with painful TMJ clicking (n = 20); patients with painless TMJ clicking (n = 20); and healthy controls (n = 20). Participants were genotyped for the following SNPs using real-time polymerase chain reaction: MMP1 -16071G/2G, COMT Val158Met, TNFα -308, IL1β +3954, IL6 -174, and IL10 -1082. The pressure pain threshold (PPT) of the TMJ was also assessed. All variables were compared among groups. Patients with painful TMJ clicking had a significant association and a higher frequency of MMP1 -16071G/2G (P = .042), COMT Val158Met (P = .030), and TNFα -308 (P = .016) when compared to the other groups, as well as a lower frequency of IL10 -1082. Considering PPT values, a progressively lower mean was found in individuals with painful TMJ clicking, followed sequentially by the painless TMJ clicking and the control groups. This pilot study showed that patients with painful TMJ clicking had a significant association with mutant genotypes related to degradation of extracellular matrix components, pain, proinflammation, and anti-inflammation. Furthermore, these patients also had significantly lower TMJ PPT values in all comparisons.

Secreted protein acidic and rich in cysteine (SPARC) and a disintegrin and metalloproteinase with thrombospondin type 1 motif (ADAMTS1) increments by the renin-angiotensin system induce renal fibrosis in deoxycorticosterone acetate-salt hypertensive rats

Secreted protein acidic and rich in cysteine (SPARC), an extracellular matrix (ECM) protein, was recently shown to induce collagen deposition through the production of a disintegrin and metalloproteinase with thrombospondin type 1 motif (ADAMTS1) in the aging heart. ADAMTS1 regulates ECM turnover by degrading ECM components, and its excessive activation contributes to various pathological states, including fibrosis. The present study investigated the pathophysiological regulation and role of SPARC and ADAMTS1 in renal fibrosis using uninephrectomized rats treated with deoxycorticosterone acetate (DOCA, 40 mg/kg/week, subcutaneously) and salt (1% in drinking water). The administration of DOCA and salt gradually and significantly elevated systolic blood pressure during the 3-week treatment period, induced proteinuria, decreased creatinine clearance, and increased NADPH oxidase-derived superoxide production, malondialdehyde concentrations, and monocyte chemoattractant protein-1 and osteopontin expression in the kidneys. Glomerulosclerosis, fibrillar collagen deposition, and transforming growth factor-β expression increased in a time-dependent manner, and SPARC and ADAMTS1 expression showed a similar pattern to these changes. The angiotensin II type-1 receptor blocker losartan suppressed the overexpression of SPARC and ADAMTS1, and an in vitro exposure to angiotensin II induced the production of both SPARC and ADAMTS1 in renal fibroblast NRK-49F cells. Knockdown of the SPARC gene with small interfering RNA reduced all forms (the 110-kDa latent and 87- and 65-kDa bioactive forms) of ADAMTS1 expression as well as collagen production. These results suggest that SPARC is induced by the renin-angiotensin system and may be a fibrogenic factor, at least in part, by producing ADAMTS1 in hypertensive renal disease.

Use of MALDI Mass Spectrometry Imaging to Identify Proteomic Signatures in Aortic Aneurysms after Endovascular Repair.

Endovascular repair (EVAR) has become the standard procedure in treating thoracic (TAA) or abdominal aortic aneurysms (AAA). Not entirely free of complications, a persisting perfusion of the aneurysm after EVAR, called Endoleak (EL), leads to reintervention and risk of secondary rupture. How the aortic wall responds to the implantation of a stentgraft and EL is mostly uncertain. We present a pilot study to identify peptide signatures and gain new insights in pathophysiological alterations of the aortic wall after EVAR using matrix-assisted laser desorption or ionization mass spectrometry imaging (MALDI-MSI). In course of or accompanying an open aortic repair, tissue sections from 15 patients (TAA = 5, AAA = 5, EVAR = 5) were collected. Regions of interest (tunica media and tunica adventitia) were defined and univariate (receiver operating characteristic analysis) statistical analysis for subgroup comparison was used. This proof-of-concept study demonstrates that MALDI-MSI is feasible to identify discriminatory peptide signatures separating TAA, AAA and EVAR. Decreased intensity distributions for actin, tropomyosin, and troponin after EVAR suggest impaired contractility in vascular smooth muscle cells. Furthermore, inability to provide energy caused by impaired respiratory chain function and continuous degradation of extracellular matrix components (collagen) might support aortic wall destabilization. In case of EL after EVAR, this mechanism may result in a weakened aortic wall with lacking ability to react on reinstating pulsatile blood flow.

Взаємозв’язок активності матриксної металопротеїнази-2 та -9 в гомогенаті міокарда зі змінами клітинного складу стінки вінцевих артерій в експерименті

Matrix metalloproteinases are involved in a complex multifactorial process of atherosclerotic plaque formation and play a leading role in the degradation of the extracellular matrix components and increase the migratory activity of cellular elements of the vascular wall. Despite a number of scientific studies, it is necessary to identify clear biochemical markers for the development of atherosclerotic lesions. The purpose of the study was to investigate the relationship between the activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 in myocardial homogenate with changes of the cells composition in the coronary arteries wall in experimental atherosclerosis. Materials and methods. The studies were performed on 76 nonlinear rats, which were divided into 3 groups: group Ia was the control (n=20) – animals, injected intracutaneously with 0.1 ml of 0.9 % sodium chloride solution; Ib – comparison group (n=20) – animals injected with incomplete Freund’s adjuvant at the dose of 0.1 ml intracutaneously and II – experimental group (n=36), which were immunized with native human low-density lipoprotein at a single dose of 200 μg, diluted in 0.1 ml of incomplete Freund’s adjuvant, regardless of the weight. The experiment lasted for 20 weeks. Material sampling was performed, starting from the 4th week after the drug administration. From the coronary arteries and the adjacent myocardium, microslides were made according to the generally accepted technique, which were stained with hematoxylin and eosin, according to the methods of Van Gizon, Mallory and Sudan III. The activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 was determined by enzyme-zimografic method. Results and discussion. The growth of matrix metalloproteinase-2 and matrix metalloproteinase-9 activity in atherosclerotic lesions showed stages of degradation of extracellular matrix components: the dynamics of matrix metalloproteinase-2 activity during the experiment increased from 109.1±1.23 % at the 12th week to 127.32±0.99 % at the 20th week. The increased activity of matrix metalloproteinase-2 was associated with an increase in the number of leukocytes and macrophages, including foam cells. The activity of matrix metalloproteinase-9 reached the highest values of 98.24±0.82% at the 8th period from the onset of changes to the final level of 86.26±0.54% at the maximum terms of the experiment. Conclusion. The growing activity of matrix metalloproteinase-9 indicated the development of early atherosclerotic lesions, while the high level of activity of matrix metalloproteinase-2 indicated significant structural changes in the wall of the coronary arteries

A Biopolymer‐delivered MMP‐2 Inhibitory for Treatment of Renal Fibrosis

Renal fibrosis, the buildup of extracellular matrix (ECM) components and scar tissue leading to organ failure, is the final common pathway of chronic kidney disease (CKD). There are currently no pharmacological treatments for CKD that specifically target the fibrosis process. Matrix metalloproteinases (MMPs) are involved in ECM homeostasis and degrade ECM components during tissue repair. Although an increase in MMPs can have anti-fibrotic effects, some MMPs can also process and activate chemokines and cytokines that are pro-inflammatory. Studies have shown that in the initial stages of CKD, there is an increase in the expression and activity of MMP-2, a gelatinase known to activate pro-TGF-β, TNF-α, and TNF- β, which are known to be pro-inflammatory and profibrotic. Furthermore, MMP-2 can promote epithelial to mesenchymal transition (EMT), a process in which epithelial cells lose their cell-to-cell adhesion and gain the ability to migrate, proliferate, and differentiate to profibrotic mesenchymal cells. Our goal is to develop an MMP-2 inhibitor that specifically targets the renal cells to reduce or reverse the effects of fibrosis. We created a chimeric fusion protein between an MMP-2 inhibitory peptide derived from beta amyloid precursor peptide (APP-IP) and an elastin like polypeptide (ELP) drug carrier. ELP is has been shown to accumulate in the kidney and has a long and tunable plasma half-life, which would allow for the therapeutic to have a sustained activity with minimal off target effects. An MMP-2 fluorometric assay revealed that the ELP – MPP-2 inhibitory peptide fusion protein (ELP-APP-IP) maintained potent MMP-2 inhibitory activity (IC50 = 1 mM, Figure panel A). Biodistribution of ELP-APP-IP or ELP control was determined in Sprague Dawley rats following bolus intravenous injection (30 mg/kg). Pharmacokinetic analysis revealed a terminal half-life approximately 90 minutes for both ELP and ELP-APP-IP. Ex vivo imaging of the organs showed that both ELP and ELP-APP-IP localized to the kidneys at levels approximately 16 times higher than in other major organs (brain, liver spleen, heart and lungs, Figure panel B,C). Histological analysis revealed that ELP and ELP-APP-IP accumulated primarily in the renal cortex (Figure panel D). A pilot efficacy study was conducted in Dahl Salt Sensitive Rats (S-Rats) on a high salt diet (8% NaCl) as a model of hypertension induced glomerulosclerosis. S-Rats (male, 12 weeks of age) on high salt diet were treated with ELP or ELP-APP-IP (n = 5 per group) for 3 weeks via osmotic minipumps (10 mg/kg/day). Urine was collected via metabolic caging 24 hours prior to initiation of treatment and high salt diet, then again weekly throughout the study. 21 days after initiation of treatment, the rats were euthanized, and blood and major organs were harvested for analysis. Rats on a high salt diet treated with ELP-APP-IP had similar urine production to rats treated with ELP. However, there was a trend for reduced onset of proteinuria in rats treated with ELP-APP-IP (two way ANOVA, treatment factor p = 0.088, Figure panel E), suggesting ELP-APP-IP has the potential to reduce kidney damage and slow the progression of renal disease.

A Combined Phytochemistry and Network Pharmacology Approach to Reveal Potential Anti-NSCLC Effective Substances and Mechanisms in Marsdenia tenacissima (Roxb.) Moon (Stem).

Marsdeniae tenacissimae Caulis is a traditional Chinese medicine, named Tongguanteng (TGT), that is often used for the adjuvant treatment of cancer. In our previous study, we reported that an ethyl acetate extract of TGT had inhibitory effects against adenocarcinoma A549 cells growth. To identify the components of TGT with anti-tumor activity and to elucidate their underlying mechanisms of action, we developed a technique for isolating compounds, which was then followed by cytotoxicity screening, network pharmacology analysis, and cellular and molecular experiments. We isolated a total of 19 compounds from a TGT ethyl acetate extract. Two novel steroidal saponins were assessed using an ultra-performance liquid chromatography-photodiode array coupled with quadrupole time-of-flight mass (UPLC-ESI-Q/TOF-MS). Then, we screened these constituents for anti-cancer activity against non-small cell lung cancer (NSCLC) in vitro and obtained six target compounds. Furthermore, a compound-target-pathway network of these six bioactive ingredients was constructed to elucidate the potential pathways that controlled anticancer effects. Approximately 205 putative targets that were associated with TGT, as well as 270 putative targets that were related to NSCLC, were obtained from online databases and target prediction software. Protein–protein interaction networks for drugs as well as disease putative targets were generated, and 18 candidate targets were detected based on topological features. In addition, pathway enrichment analysis was performed to identify related pathways, including PI3K/AKT, VEGF, and EGFR tyrosine kinase inhibitor resistance, which are all related to metabolic processes and intrinsic apoptotic pathways involving reactive oxygen species (ROS). Then, various cellular experiments were conducted to validate drug-target mechanisms that had been predicted using network pharmacology analysis. The experimental results showed the four C21 steroidal saponins could upregulate Bax and downregulate Bcl-2 expression, thereby changing the mitochondrial membrane potential, producing ROS, and releasing cytochrome C, which finally activated caspase-3, caspase-9, and caspase-8, all of which induced apoptosis in A549 cells. In addition, these components also downregulated the expression of MMP-2 and MMP-9 proteins, further weakening their degradation of extracellular matrix components and type IV collagen, and inhibiting the migration and invasion of A549 cells. Our study elucidated the chemical composition and underlying anti-tumor mechanism of TGT, which may be utilized in the treatment of lung cancer.