Quantification of Extracellular Matrix Proteins from a Rat Lung Scaffold to Provide a Molecular Readout for Tissue Engineering

The use of extracellular matrix (ECM)¹ scaffolds, derived from decellularized tissues for engineered organ generation, holds enormous potential in the field of regenerative medicine. To support organ engineering efforts, we developed a targeted proteomics method to extract and quantify extracellular matrix components from tissues. Our method provides more complete and accurate protein characterization than traditional approaches. This is accomplished through the analysis of both the chaotrope-soluble and -insoluble protein fractions and using recombinantly generated stable isotope labeled peptides for endogenous protein quantification. Using this approach, we have generated 74 peptides, representing 56 proteins to quantify protein in native (nondecellularized) and decellularized lung matrices. We have focused on proteins of the ECM and additional intracellular proteins that are challenging to remove during the decellularization procedure. Results indicate that the acellular lung scaffold is predominantly composed of structural collagens, with the majority of these proteins found in the insoluble ECM, a fraction that is often discarded using widely accepted proteomic methods. The decellularization procedure removes over 98% of intracellular proteins evaluated and retains, to varying degrees, proteoglycans and glycoproteins of the ECM. Accurate characterization of ECM proteins from tissue samples will help advance organ engineering efforts by generating a molecular readout that can be correlated with functional outcome to drive the next generation of engineered organs.Organ transplantation is an established, lifesaving therapy for patients with chronic end-stage diseases. However, transplantation as a therapeutic option is limited by availability of suitable donor organs (1). Although advancements in surgical techniques, such as successful implementation of bilateral lung transplants and improved immunosuppressant treatments, have led to more successful outcomes in recent years, the percentage of people that die while on the transplant wait list has increased (2, 3). One attractive approach to meet this demand is the in vitro generation of organs using decellularized tissues as scaffolds for recellularization. For complex organs such as the lung, these tissue scaffolds can be derived from a donor organ that would have otherwise been unfit for transplantation. This whole organ scaffold can be recellularized using a patient''s own primary or stem-derived cells, thus eliminating many issues related to graft/host incompatibility. This approach was recently used to generate lungs that, when implanted in rat recipients, allowed for gas exchange (4, 5). However, examination of the lung indicated leakage of erythrocytes into the alveolar space, indicating a compromised capillary-endothelial barrier. These exciting results highlighted the potential of the method for organ transplantation but also the need for improved molecular readouts to guide engineering efforts.Efficient reseeding of decellularized scaffolds has been shown to be dependent on retaining native ECM structural integrity and elasticity (6). Local variations in expression of abundant proteins in the ECM scaffolding (collagens, laminins, fibronectins) have been correlated to variance in cell repopulation and subsequent proliferation (7). It is thought that retaining specific ECM components and architectures may allow cells to be directed back to a tissue-specific niche during reseeding and that small changes in abundance of these molecular cues can drastically affect the recellularization process (8). Current methods used to characterize the protein composition of native and acellular tissues involve antibody- or dye-based staining, hydroxyproline assays assessing collagen content, or relative quantification of proteins by liquid chromatography tandem mass spectrometry (LC-MS/MS) (9, 10). All of these methods either fall short in specificity, accurate quantification, or both. A more complete and accurate method for protein characterization would provide a valuable tool for tissue engineering efforts, while shedding light on the possible molecular mechanisms resulting in cell seeding variability and alterations in mechanical properties of engineered lung tissues.Current relative quantification strategies (iTRAQ, Spectral Counting, dimethyl labeling, others) (11–15) perform well when the majority of protein in samples does not change, there are approximately equal increases and decreases in protein levels, or in cases where proteins that are known not to change in abundance can be used for normalization. However, normalization steps often employed have the potential to introduce experimental bias (16). The decellularization process differentially removes and enriches proteins in the ECM scaffolding, depleting some proteins with high efficiency while leaving others mostly intact. This makes relative comparisons between native and decellularized lung challenging. Although strategies can be employed in an attempt to normalize data (17), there is a distinct advantage to quantification methods using stable isotope labeled (SIL) peptides in this application. Here, we developed ECM targeted, isotopically labeled peptides using the QconCAT approach first described by Beynon et al. (18). SIL quantification allows for intra- and intersample comparison of heterogeneous tissues, such as native organs and decellularized scaffolds, with high accuracy and precision.The ECM is largely responsible for defining the biomechanical properties of organs. Maintaining structural rigidity and native microarchitecture through the decellularization process makes an acellular organ a good candidate to serve as a tissue scaffold (19, 20). These same characteristics are a central reason why the ECM is challenging to characterize using common bottom-up proteomics approaches (21). Currently accepted and widely used digestion methods require proteins to be solubilized for bottom-up proteomic analysis (22). Recent papers have reported characterization of the ECM fraction from tissues through the use of strong chaotropes (11, 21, 23–27) or cellular fractionation followed by strong detergent (10, 28, 29). However, in our experience, these protocols invariably yield various sizes of an insoluble protein-containing pellet when applied to a variety of tissue samples (heart, lung, and mammary gland). On one end of the spectrum, methods utilizing deglycosylation and enzymatic digestions for clarification of partial solubilized protein slurries yields good ECM coverage with a high number of spectral matches for collagen alpha-1(I), a highly abundant ECM protein in lung (28). On the other end of the spectrum, methods using only detergents or chaotropes for solubilization result in protein pellets that are generally removed prior to LC-MS/MS analysis. These pellets often contained a majority of fibrillar proteins, resulting in quantitative errors. Consistent with this finding, several of these studies characterizing tissue engineered lungs do not report the identification of collagen alpha-1(I) (8, 10, 30). We believe these observations result from a failure to solubilize and enzymatically digest insoluble ECM proteins. To this end, we explored the use of chemical digestion of the insoluble pellet to improve coverage of the ECM proteome from tissue. This method has been used to quantify protein levels from native and decellularized lungs to determine decellularization specificity and efficiency. The accurate characterization of ECM proteins from lung samples should advance tissue engineering efforts by yielding a readout that can be correlated with functional outcome to drive further development.     

Menstrual cycle characterization and artificial insemination in the black mangabey (Cercocebus aterrimus)

Concentrations of immunoreactive estrone conjugates, pregnanediol-3-glucuronide, and luteinizing hormone were measured and indexed to creatinine in daily urine samples from three female black mangabeys (Cercocebus aterrimus). Daily observations of menstruation and perineal tumescence were recorded. The mean ± SEM lengths of the menstrual cycle [apparent cycle length of 26.0 ± 0.8 days determined by observation of intermenstrual intervals (n = 26); physiologic cycle length of 31.3 ± 5 days determined by urinary endocrine analysis (n = 4)], follicular phase [16.5 ± 4 days (n = 4)], and luteal phase [14.8 ± 1 day (n = 4)] were determined. The apparent cycle length is probably more accurate. Perineal tumescence began during or shortly after menstruation, increased concomitantly with increasing follicular phase conjugated estrone values, and reached maximal size in the periovulatory period. Ovulation was closely followed by a drop in conjugated estrone levels, an increase in urinary pregnanediol-3-glucuronide, and perineal detumescence. Peak concentrations of conjugated estrone and luteinizing hormone values were coincident. Pregnanediol-3-glucuronide accurately reflected luteal function in the black mangabey. Knowledge of the menstrual cycle parameters and their correlation to perineal tumescence was used to time artificial inseminations. Semen was obtained by rectal electroejaculation. Coagulum and extended semen, or trypsin-digested coagulum, were used for insemination. One insemination of trypsin-digested coagulum at the external os of the cervix resulted in a probable conception, follówed by apparent abortion after 3 weeks.     

The Quantitative Relationship Between “The Metabolic Syndrome” and Abdominal Obesity in Women

Abdominal obesity is closely associated with the presence of metabolic risk factors and elevated blood pressure in selected materials. This has, however, never been analyzed quantitatively in a non-selected cohort. Therefore, in a population-based study of 1462 Swedish women, four selected risk factors for non-insulin dependent diabetes mellitus (NIDDM) and cardiovascular disease (CVD), serum triglycerides, blood glucose and systolic blood pressure and also serum insulin in a subsample, were examined in relation to regional and overall obesity. This was performed by subdividing the age adjusted sample into quintiles of waist to hip circumference ratio (WHR) or body mass index (BMI) as indicators of abdominal distribution of body fat and overall obesity, respectively. The risk factors serum triglycerides, blood glucose, blood pressure and serum insulin were defined as being elevated when the value of the risk factor was higher than the mean plus one or two standard deviations of the total age-adjusted cohort. The percentage of women with elevated risk factors according to this definition was then calculated in each of these quintiles. Having a risk factor which was elevated according to the definition was significantly correlated to WHR and BMI (p<0.0001) independent of age. The presence of one or several of these elevated risk factors was clearly higher than expected in the fifth quintile of WHR, and to a lesser extent in the fifth quintile of BMI while this was not the case in the lower quintiles of WHR and BMI. When studying the combination of the WHR and BMI, the presence of risk factors higher than the mean plus two standard deviations increased gradually with WHR in all five quintiles of BMI. A significant association was observed between WHR and presence of risk factors independent of BMI (p<0.0001) but BMI did not remain significantly correlated to presence of risk factors when controlling for WHR (p=0.09). These results indicate that abdominal distribution of body fat in women independently of general obesity is closely associated with metabolic risk factors including elevated blood pressure, a metabolic syndrome with increased risk for cardiovascular disease and non-insulin dependent diabetes mellitus.     

Dietary Intake in Relation to Restrained Eating,Disinhibition, and Hunger in Obese and Nonobese Swedish Women

The aims of this study were to: describe dietary intakes of obese and nonobese middle-aged women using a validated food frequency questionnaire; to assess dietary restraint, disinhibition, and hunger by the three factor eating questionnaire (TFEQ) in obese and nonobese samples and determine which of the factors are independently associated with obesity; and to examine correlations between selected nutritional variables and the TFEQ factors. Subjects studied included 179 obese Swedish women (BMI>32) and 147 nonobese population-based controls (BMI<28). Age-adjusted mean energy intake was significantly higher in obese women (2730 ± 78 vs. 2025 ± 85 kcal, p<0.0001). In absolute and relative terms, fat intake was higher and alcohol intake was lower in the obese subjects. Disinhibition was the strongest TFEQ factor independently differentiating the obese and nonobese states, i.e., after adjustment for restraint and hunger. Within the obese sample, strong associations were seen between energy intake and disinhibition (p=0.0005) and hunger (p=0.0004). The association between energy intake and restrained eating was negative and weaker (p=0.04). No such associations were seen in nonobese women. Thus, using a dietary instrument that is valid and unbiased with respect to obesity, strong psychological correlates, possibly causal, of variability in energy intake were detected in middle-aged women with obesity. Disinhibition is associated with both obesity and high-energy intakes and is therefore an important factor to consider in the treatment of women with obesity.     

An isocoumarin and other phenolic components of Ononis natrix

From the n-hexane extract of the whole aerial part of flowering Ononis natrix, besides homopterocarpin and some known terpenoids and sterols, three new compounds: 8-hydroxy-6-methoxy-3-undecyl-3,4-dihydroisocoumarin, 5-(2-acetoxytridecyl)-3-methoxyphenol and 5-(2-hydroxytridecyl)-3-methoxyphenol have been isolated.     

Effect of streptozotocin at the insulin pre-receptor, receptor and post-receptor level in adipocytes of rats

The relationship among plasma insulin disappearance, insulin binding to specific receptors in fat cells and antilipolytic insulin activity in streptozotocin-diabetic rats has been studied. Male Wistar rats were injected streptozotocin (65 mg/kg body weight) or saline by cardiac puncture. Decreased insulin levels and increased insulin degradation together with an increase in insulin binding were found in diabetic rats. The increase in insulin binding was related to an increase in the number of insulin receptors rather than to a change in receptor affinity. These findings at the pre-receptor and receptor levels could be correlated with an increase in antilipolytic insulin activity. However our results suggest that the mechanism for insulin action occurred through a potentiation of the norepinephrine lipolytic activity.     

Adrenergic receptors are not mediators in the lipolytic effect of somatostatin in rat adipocytes

Beta-adrenergic receptors have been purported to act as possible mediators in the lipolytic effect of somatostatin in vivo. Investigations with isolated rat adipocytes studying the lipolytic activity of somatostatin (1.7 x 10(-7) M), glucagon (8.1 x 10(-8 M) and norepinephrine (10(-6) M), have shown that the lipolytic effect stimulated by somatostatin is not altered by 10(-5) M propranolol (beta-antagonist); is significantly enhanced by 10(-5) M isoproterenol (beta-agonist) and is not altered by the addition of 10(-6) M phenoxybenzamine (alpha-antagonist) or 10(-6) M phenylephrine (alpha-agonist). Similar results were found when lipolysis was stimulated by glucagon, whereas the lipolytic effect stimulated by norepinephrine was blocked by propranolol. These results indicate that the direct lipolytic effect of somatostatin on isolated rat adipocytes does not seem to be mediated through mechanisms involved with adrenergic receptors.     

The effect of substituted phenothiazines on the mutagenicity of benzo[a]pyrene

The effect of phenothiazine and 11 of its derivatives on the mutagenicity of benzo[a]pyrene, as measured by the Ames test was investigated. Significant anti-mutagenic activity was detected for 10 phenothiazine derivatives, with the 2-chloro derivative being the most effective inhibitor tested and promazine the only phenothiazine drug tested which has no demonstrable inhibitory activity. It is considered that the anti-mutagenic activity and therefore potentially anticarcinogenic activity of these derivatives should be of interest to epidemiologists.