Tissue Protease Activity Research Articles

Role of Kallikrein 7 in Body Weight and Fat Mass Regulation.

Increased plasma and adipose tissue protease activity is observed in patients with type 2 diabetes and obesity. It has been proposed that specific proteases contribute to the link between obesity, adipose tissue inflammation and metabolic diseases. We have recently shown that ablation of the serine protease kallikrein-related peptidase 7 (Klk7) specifically in adipose tissue preserves systemic insulin sensitivity and protects mice from obesity-related AT inflammation. Here, we investigated whether whole body Klk7 knockout (Klk7−/−) mice develop a phenotype distinct from that caused by reduced Klk7 expression in adipose tissue. Compared to littermate controls, Klk7−/− mice gain less body weight and fat mass both under chow and high fat diet (HFD) feeding, are hyper-responsive to exogenous insulin and exhibit preserved adipose tissue function due to adipocyte hyperplasia and lower inflammation. Klk7−/− mice exhibit increased adipose tissue thermogenesis, which is not related to altered thyroid function. These data strengthen our recently proposed role of Klk7 in the regulation of body weight, energy metabolism, and obesity-associated adipose tissue dysfunction. The protective effects of Klk7 deficiency in obesity are likely linked to a significant limitation of adipocyte hypertrophy. In conclusion, our data indicate potential application of specific KLK7 inhibitors to regulate KLK7 activity in the development of obesity and counteract obesity-associated inflammation and metabolic diseases.

Prevalence and Predictors of Urinary Incontinence at 1 Year Postpartum.

Postpartum urinary incontinence estimates range from 13% to 47%. Clinical factors associated with incontinence 1 year after first delivery are varied. We assessed the prevalence of and factors associated with urinary incontinence in primiparous women at 12 months postpartum. Ancillary analysis of 99 nulliparous women from a prospective cohort study that assessed participants during the first and third trimesters and 12 months postpartum. Our primary outcome was urinary incontinence 12 months postpartum. Women were asked "How often do you experience urine leakage?" and considered to have urinary incontinence if a response other than "never" was reported. We collected vaginal swabs for assessment of matrix metalloproteinase-9 activity, a measure of tissue remodeling. Bivariable and logistic regression analyses were used to compare women with and without postpartum urinary incontinence. Of 99 primiparous women, 55% (n = 54) reported urinary incontinence at 12 months postpartum. Logistic regression demonstrated that urinary incontinence during pregnancy (odds ratio, 34.3; 95% confidence interval, 7.9-149.2) and a decrease in matrix metalloproteinase 9 activity between the first and third trimesters (odds ratio, 19.34; 95% confidence interval, 3.47-107.84) were associated with postpartum urinary incontinence. The sensitivity and specificity of urinary incontinence during pregnancy for predicting postpartum urinary incontinence were 87% and 67%, respectively. The positive and negative predictive values were 76% and 81%, respectively. Urinary incontinence affected 55% of primiparous women at 12 months postpartum. Urinary incontinence during pregnancy was strongly associated with postpartum incontinence. Importantly, vaginal tissue protease activity during pregnancy represents a possible mechanism for and biomarker of postpartum urinary incontinence.

Enterococcus faecalis exploits the human fibrinolytic system to drive excess collagenolysis: implications in gut healing and identification of druggable targets.

Perforations, anastomotic leak, and subsequent intra-abdominal sepsis are among the most common and feared complications of invasive interventions in the colon and remaining intestinal tract. During physiological healing, tissue protease activity is finely orchestrated to maintain the strength and integrity of the submucosa collagen layer in the wound. We (Shogan, BD et al. Sci Trans Med 7: 286ra68, 2015.) have previously demonstrated in both mice and humans that the commensal microbe Enterococcus faecalis selectively colonizes wounded colonic tissues and disrupts the healing process by amplifying collagenolytic matrix-metalloprotease activity toward excessive degradation. Here, we demonstrate for the first time, to our knowledge, a novel collagenolytic virulence mechanism by which E. faecalis is able to bind and locally activate the human fibrinolytic protease plasminogen (PLG), a protein present in high concentrations in healing colonic tissue. E. faecalis-mediated PLG activation leads to supraphysiological collagen degradation; in this study, we demonstrate this concept both in vitro and in vivo. This pathoadaptive response can be mitigated with the PLG inhibitor tranexamic acid (TXA) in a fashion that prevents clinically significant complications in validated murine models of both E. faecalis- and Pseudomonas aeruginosa-mediated colonic perforation. TXA has a proven clinical safety record and is Food and Drug Administration approved for topical application in invasive procedures, albeit for the prevention of bleeding rather than infection. As such, the novel pharmacological effect described in this study may be translatable to clinical trials for the prevention of infectious complications in colonic healing.NEW & NOTEWORTHY This paper presents a novel mechanism for virulence in a commensal gut microbe that exploits the human fibrinolytic system and its principle protease, plasminogen. This mechanism is targetable by safe and effective nonantibiotic small molecules for the prevention of infectious complications in the healing gut.

Loss of skin elasticity is associated with pulmonary emphysema, biomarkers of inflammation, and matrix metalloproteinase activity in smokers

BackgroundElastin breakdown and the resultant loss of lung elastic recoil is a hallmark of pulmonary emphysema in susceptible individuals as a consequence of tobacco smoke exposure. Systemic alterations to the synthesis and degradation of elastin may be important to our understanding of disease phenotypes in chronic obstructive pulmonary disease. We investigated the association of skin elasticity with pulmonary emphysema, obstructive lung disease, and blood biomarkers of inflammation and tissue protease activity in tobacco-exposed individuals.MethodsTwo hundred and thirty-six Caucasian individuals were recruited into a sub-study of the University of Pittsburgh Specialized Center for Clinically Orientated Research in chronic obstructive pulmonary disease, a prospective cohort study of current and former smokers. The skin viscoelastic modulus (VE), a determinant of skin elasticity, was recorded from the volar forearm and facial wrinkling severity was determined using the Daniell scoring system.ResultsIn a multiple regression analysis, reduced VE was significantly associated with cross-sectional measurement of airflow obstruction (FEV1/FVC) and emphysema quantified from computed tomography (CT) images, β = 0.26, p = 0.001 and β = 0.24, p = 0.001 respectively. In emphysema-susceptible individuals, elasticity-determined skin age was increased (median 4.6 years) compared to the chronological age of subjects without emphysema. Plasma biomarkers of inflammation (TNFR1, TNFR2, CRP, PTX3, and SAA) and matrix metalloproteinase activity (MMP1, TIMP1, TIMP2, and TIMP4) were inversely associated with skin elasticity.ConclusionsWe report that an objective non-invasive determinant of skin elasticity is independently associated with measures of lung function, pulmonary emphysema, and biomarkers of inflammation and tissue proteolysis in tobacco-exposed individuals. Loss of skin elasticity is a novel observation that may link the common pathological processes that drive tissue elastolysis in the extracellular matrix of the skin and lung in emphysema-susceptible individuals.

Spatial Distribution of Endogenous Tissue Protease Activity in Gastric Carcinoma Mapped by MALDI Mass Spectrometry Imaging.

Aberrant protease activity has been implicated in the etiology of various prevalent diseases including neurodegeneration and cancer, in particular metastasis. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) has recently been established as a key technology for bioanalysis of multiple biomolecular classes such as proteins, lipids, and glycans. However, it has not yet been systematically explored for investigation of a tissue's endogenous protease activity. In this study, we demonstrate that different tissues, spray-coated with substance P as a tracer, digest this peptide with different time-course profiles. Furthermore, we reveal that distinct cleavage products originating from substance P are generated transiently and that proteolysis can be attenuated by protease inhibitors in a concentration-dependent manner. To show the translational potential of the method, we analyzed protease activity of gastric carcinoma in mice. Our MSI and quantitative proteomics results reveal differential distribution of protease activity - with strongest activity being observed in mouse tumor tissue, suggesting the general applicability of the workflow in animal pharmacology and clinical studies.

Serine Protease Inhibition Reduces Post-Ischemic Granulocyte Recruitment in Mouse Intestine

Proteases and proteinase-activated receptor (PAR) activation are involved in several intestinal inflammatory conditions. We hypothesized that serine proteases and PAR activation could also modulate the intestinal injury induced by ischemia-reperfusion (I-R). C57Bl/6 mice were subjected to 90 minutes of intestinal ischemia followed or not by reperfusion. Sham-operated animals served as controls. After ischemia, plasma and tissue serine protease activity levels were increased compared to the activity measured in plasma and tissues from sham-operated mice. This increase was maintained or further enhanced after 2 and 5 hours of reperfusion, respectively. Trypsin (25 kDa) was detected in tissues both after ischemia and 2 hours of reperfusion. Treatment with FUT-175 (10 mg/kg), a potent serine protease inhibitor, increased survival after I-R, inhibited tissue protease activity, and significantly decreased intestinal myeloperoxidase (MPO) activity and chemokine and adhesion molecule expression. We investigated whether serine proteases modulate granulocyte recruitment by a PAR-dependent mechanism. MPO levels and adhesion molecule expression were significantly reduced in I-R groups pre-treated with the PAR(1) antagonist SCH-79797 (5 mg/kg) and in Par(2)(-/-)mice, compared, respectively, to vehicle-treated group and wild-type littermates. Thus, increased proteolytic activity and PAR activation play a pathogenic role in intestinal I-R injury. Inhibition of PAR-activating serine proteases could be beneficial to reduce post-ischemic intestinal inflammation.

Parameters of energy and nitrogen metabolism in rats under insulin-induced hypoglycemia

Repeated severe insulin-induced hypoglycemia in rats has led to an increase in aminotransferase, glutaminase, and glutamate dehydrogenase activities in the liver; protease activities in tissues; and in blood serum levels of free fatty acids, urea, and uric acid. These changes are indicative of gluconeogenesis activation in animals exposed to hyperinsulinization. Decreased rates of glycolysis and glycogenolysis, reduced activities of NADP-dependent dehydrogenases, and substantial changes in the activities of enzymes responsible for metabolism of nucleotides and transmitter amino acids have been observed in the brain. All these changes are mainly associated with hypoglycemia and activation of the contrainsular system and can play a significant role in pathogenesis of posthypoglycemic encephalopathy.

Effect of diet on tissue protease activity.

Rats 1, 3, 12, and 24 months old were fed diets low in protein (8% casein), and proteolytic activity in tissue from brain, liver, and lung was determined. After a low-protein diet was fed for 4 weeks to 1-month-old rats, there was a significant increase in cathepsin D activity in liver, and calpain activity was increased in lung. Little change was seen in proteolytic activity in brain. In 12-month-old rats, there was an increase in cathepsin D activity in brain and liver. In 24-month-old rats, cathepsin D activity in the liver and calpain activity in lung were increased. There was no change in proteolytic activity in the brain. When animals were fed diets supplemented with fatty acids or antioxidants for 2 months, in 3-month-old rats calpain activity was increased in brain but decreased in lung. Cathepsin D activity was significantly increased in young and adult animals in brain and in liver. These observations suggest that diet changes result in significant alteration in tissue calpain and cathepsin D levels, and possibly activity, in vivo. Generally, changes are greater for cathepsin D than for calpain, and are smaller in brain than in other tissues.

ANALYSIS OF BREAST-TISSUE CATHEPSIN-D ISOFORMS FROM PATIENTS WITH BREAST-CANCER, BENIGN BREAST DISEASE AND FROM NORMAL CONTROLS

The isoform composition of cathepsin D has been investigated by isoelectric focusing of breast tissue supernatant fluids from patients with breast cancer, benign breast disease and from normal controls. The results indicated the presence of several acid protease isoforms between pi values of 2 to 8. Three of these isoforms (with approximate pIs of 6.0, 6.4 and 7.0) were pepstatin-inhibitable but not inhibitable by a mixture of protease inhibitors for seryl, cysteinyl and metalloproteases. These three isoforms, and not the more acidic isoforms, contained a 31 kD protein band which was recognized by polyclonal antibodies against cathepsin D, suggesting that these isoforms are cathepsin D. Further evidence that these isoforms are cathepsin D came from studies in which the pepstatin-inhibitable protease activity and not the pepstatin-uninhibitable protease activity, bound to and was elutable from, an immunoaffinity resin made by coupling anticathepsin D polyclonal antibodies to agarose. The mean relative percentage of the total breast tissue protease activity associated with pepstatin-inhibitable activity (i.e. cathepsin D) was significantly increased (p<0.01) in five breast cancer isoform profiles (64+/-4%, mean+/-S.D.) when compared to five normal control breast profiles (32+/-5%). An analysis of the three cathepsin D isoforms between pIs 6 to 7 indicated a trend of increased relative amounts of the most acidic isoform in the breast cancer isoform profiles when compared to isoform profiles from benign breast disease and normal control breast tissues. Evidence that the more acidic cathepsin D isoforms are related to the more neutral isoforms by sialylation came from studies in which neuraminidase treatment of breast tissue supernatant fluids led to decreased amounts of the most acidic isoform with a concomitant increase in the more neutral isoforms. The apparent increased relative amounts of the most acidic cathepsin D isoform in malignant breast tissue, coupled with the neuraminidase treatment results, provide further evidence that malignant breast tissue cathepsin D is abnormally glycosylated.

Enhanced interpretation of tissue protease activity by use of photographic color film as a substrate.

The enzymatic digestion of the first colored layer of gelatin in a photographic color-film processed unexposed produce a transparent colored site. Thus the use of a color-film for the histochemical localization of protease activity has proved to be an improvement over the gelatin-silver-film use. The three pigmented layers of the reversible Ferrania 3M color film, 135/36 daylight type, DIA 28 are yellow, magenta and red, and each is only 7 μ thick. Before use, the film is dampened with 0.15 M phosphate buffer pH 7.6 until the gelatin swells and placed in an incubator where the atmosphere is saturated with water vapor. Tissues, fixed in 4% formaldehyde-saline at 4 C for 24–48 hr, are cut at 10–20 μ on a freezing microtome. Sections are mounted as soon as possible on color-film and incubated. No free fluid should be left over the sections. After incubation the film is allowed to dry naturally and then protected with a cover glass applied with Canada balsam. It was possible to ascertain proteolytic activity in the digestive apparatus of the pigeon embryo on the 14th day of incubation, at the same time as the zymogen granules became visible through the electron microscope.