Chronic asthma and Mesenchymal stem cells: Hyaluronan and airway remodeling

NIH 3 T3 murine fibroblast cells (ATCC CRL-1658) were grown in specialized DMEM high glucose media (Gibco, cat #11885–092) containing 10% heat-inactivated FCS (Life Technologies, cat #16010–159), and 1% Penicillin-Streptomycin-Glutamine (PSG) (Gibco, cat #10378–016). Mesenchymal stem cells (MSCs) were received in collaboration with The Skeletal Research Center at Case Western Reserve University in Cleveland, OH and were grown as previously described elsewhere [11]. Briefly, the MSCs were grown in DMEM containing 10% fetal bovine serum from highly selected sources [26]. Within 14 days, the colony-forming units that were selectively expanded to near confluence were lifted from the culture dish using trypsin and re-plated at 1:3 to insure that they maintain active cell division.

cells were be mechanically removed from culture plates using plunger from 1 mL syringe, centrifuged at 1800 rpm for 9 min, and supernatant and cell pellet will be stored separately at −80 °C as previously described by Jainchill and colleagues [27].

NIH 3 T3 murine fibroblast cells were grown as previously described above. When 3 T3 fibroblast cells were 80% confluent, cells with and without stimulation had their growth medium changed to medium obtained from hMSCs.

3 T3 murine fibroblasts were lysed with RiboZol reagent (Fisher Scientific, cat #50–751-7365) and washed with chloroform (Fisher Scientific, cat # BP 1145-1 L). The lysed cells were centrifuged at 12,000 g for 15 min at 5 °C to achieve phase separation, and the RNA-containing aqueous layer was removed. The RNA was precipitated using isopropanol, centrifuged at 12,000 g for 10 min at 5 °C, washed three times with 70% ethanol, centrifuged at 7500 g for 5 min at 5 °C, and dissolved in sterile nuclease-free water (Fisher Scientific, cat # BP2484–100) at 55 °C. The cDNA was synthesized using the qScript cDNA kit (VWR, cat #101414–098) and stored at −20 °C or used immediately for Real Time-PCR (RT-PCR). RNA and cDNA concentration will be determined by spectrophotometry (NanoDrop ND-1000, Thermo Fisher). cDNA was diluted in sterile nuclease-free water to which Taqman Universal PCR Master Mix (Life Technologies, cat #4304437) and PCR primers (Life Technologies) were added. Samples were plated in a 96-well reaction plate (Applied BioSystems, cat #4306737). The reaction was run on an Applied BioSystems 7300 Real Time PCR System and analyzed using ABI 7300 Prism software and Microsoft Excel. Data was normalized to mouse GAPDH.

The snap-frozen lungs were thawed and washed twice in sterile PBS with 2 complete tablets of proteinase/protease inhibitor (Life Sciences, cat # 04693116001). The lungs were placed in plastic tubes with 700uL CHAPS buffer (50 mM Tris-HCl buffer, pH 7.4 + 150 mM NaCl +10 mM CHAPS) and 700uL PBS, and homogenized for 2 min. The supernatant was split into three 1.5 mL Eppendorf tubes (one for hydroxyproline assay, one for Sirius Red Assay, and one for RT-PCR assay). The hydroxyproline tube was centrifuged in speed vacuum for 45 min until all liquid is evaporated. Following vacuum spin, 2 mL of 6 N HCl was added to each tube and covered; place in dry bath at 110 °C for 24 h. The following day, the glass tubes were returned to the speed vacuum for 3 h, then allowed to cool at room temperature. Once cooled, 300uL of citrate/acetate buffer (5 g citric acid +1.2 mL glacial acetic acid +7.24 g sodium acetate +3.4 g NaOH) was placed in each sample tube, and filtered into another 1.5 mL Eppendorf tube, and 1 mL Chloramine T solution (0.282 g Chloramine T + 2 mL n-propanol +2 mL distilled water +16 mL citrate/acetate buffer) was added. This mixture was incubated at room temperature for 20 min. Following this incubation period, 1 mL Ehrlich’s solution (4.6 g 4-(dimethyalamino)-benzaldehyde +18.6 mL n-propanol +7.8 mL 70% perchloric acid) was added to each sample and incubated for 20 min at 65 °C. The sample were allowed to cool for 10 min at room temperature after which 200uL of standard stock solutions (hydroxyproline and citrate/acetate buffer) were added to the 96-well plate with 200uL of each sample evaluated in a spectrophotometer at 540 nm.

One-third of whole lung homogenate was placed in 1.5 mL Eppendorf tube. The tube was centrifuged at 10,000 g for 10 min. Subsequently, the resultant supernatant was placed in 100uL aliquots. Ten microliters of sample were removed and plated into 96-well plate with 140uL PBS. The plate was dried overnight at 37 °C. The following day, the plate was washed with 200uL/well of distilled water three times, after which 150uL of Sirius Red Stain (Sigma Aldrich, cat # 365548-25G) was added to each well. The plate was incubated at room temperature on a rocker for 1 h after which the plate was washed again four times with 200 uL/well of acidified water (5% acetic acid). After washing with acidified water, 150uL of NaOH (0.1 M NaOH) was added to each well and incubated at room temperate for 30 min on a rocker. After transferring to a new 96-well plate, the samples were evaluated in a spectrophotometer at 550 nm. Insoluble and soluble collagen was be measured by colorimetric assays (QuickZyme Biosciences).

To determine the state and quantity of HA, lung homogenates were processed using FACE [29]. The process involves isolating and breaking down the HA into its component disaccharides which are fluorescently labeled and then processed with gel electrophoresis and digital imaging.

Differentiating low molecular weight vs. high molecular weight HA was determined using HA analysis by agarose gel electrophoresis or HSAE. This technique is used for qualitative analysis of the size of the HA chains using proteases and nucleases to remove proteins and nucleic acid from the same leaving behind the glycosaminoglycans (GAGs) which are subsequently separated by size on agarose gel and then stained for HA. The HA size was determined by comparing to a ladder of purified HA with known molecular weight. Low molecular weight HA is <500 kDa, and high molecular weight HA is >500 kDa. Additionally, the lung homogenates were evaluated for HA synthase gene expression analyzing mRNA expression using Taqman technique as described above.

Using the ovalbumin model of asthma, the impact on hMSCs on ECM generated in response to allergic challenge was monitored by the change in trichrome staining. Trichrome stains the collagen components, reflected in bright blue staining in Fig. 2. The amount of trichrome staining was quantified by our ImagePro Program [28]. Since the impact of the stem cell therapy could be very site specific, imaging was done on both left and right lung lobes, slicing through the large and small airways (n = 5, animals per experiment, 3 different experiments). Visually, an example of the model and enhanced ECM with challenge and the quantification are shown in Fig. 2. The OVA challenge for 8 weeks enhanced ECM production relative to the saline control (Fig. 2a vs. b) as evidenced by the increased amount of bright blue staining, documented by the ImagePro analysis. To evaluate the impact of hMSC on ECM as measured anatomically, mice were treated with 10⁶ hMSC through the retro-orbital sinus as described previoiusly [11]. Visually the amount of Trichrome staining decreased in the hMSC treated groups (Fig. 2c , n = 5 animals per experiment, 3 different experiments). Using the same ImagePro program, several sections of the lung tissue, were evaluated for changes in ECM deposition. In each of the three different experiments, the administration of hMSCs decreased quantitative levels of ECM from the amount of ECM that would be present in the sham treated- OVA challenged animals (Fig. 2c).

To determine the effect on structural components of the lungs we chose to evaluate the amount of ECM deposition in our chronic asthma model using a monitor of total soluble and insoluble collagen in mice sensitived and challenged with OVA. Soluble and insoluble collagen measues in complex in vivo tissues can be a good monitor of therapeutic impact. Insoluble collagen is associated with increased tissue collagen in the structural component of the lungs. The soluble collagen levels are associated with initiation of repair against remodeling. The animal model was evaluated with or without the infusion of hMSCs. The insoluble and soluble collagen was measured by hydroxyproline and Sirius Red colorimetric assays, respectively. In Fig. 3a, OVA sensitized mice had increased amount of soluble collagen when measured using Sirius Red tissue staining and this collagen deposition was decreased in mice injected with hMSCs. In Fig. 3b, insoluble collagen deposition was measured using hydroxyproline assays. The amount of insoluble collagen in the lungs of OVA sensitized mice was increased when compared to controls, and this deposition was decreased in mice infused with hMSCs. These studies demonstrate hMSC infusion decreased insoluble and soluble ECM deposition in the form of collagen.

Previous studies have established that elevated hyaluronan (HA) in the lung is associated with inflammation in asthma [30]. There are several methods to monitor shifts in hyaluronan in tissue. FACE technology is useful in complex tissues since it allows for semi-quantification of total HA, as well as the strunctural identiy of the HA regarding the change in TSG-6 mediated glycosylation and HA converting from low molecular weight to high molecular forms which are pathogenic [29, 30]. HSAE technology, however, is able to qualitatively detect differences in size to distinguish high molecular weight vs. low molecular weight hyaluronan. As previously demonstrated in the literature [30], OVA sensitization and challenge statistically increased HA in the lungs (Fig. 4a, n = 3, p < 0.05). hMSCs infusion significantly decreased HA (Fig. 4b, n = 3, p < 0.05).