Static mechanical stretching accelerates lipid production in 3T3-L1 adipocytes by activating the MEK signaling pathway.

Understanding mechanotransduction in adipocytes is important for research of obesity and related diseases. We cultured 3T3-L1 preadipocytes on elastic substrata and applied static tensile strains of 12% to the substrata while inducing differentiation. Using an image processing method, we monitored lipid production for a period of 3-4 wk. The ratio of %-lipid area per field of view (FOV) in the stretched over nonstretched cultures was significantly greater than unity (P < 0.05), reaching ∼1.8 on average starting from experimental day ∼10. The superior coverage of the FOV by lipids in the stretched cultures was due to significantly greater sizes of lipid droplets (LDs) with respect to nonstretched cultures, starting from experimental day ∼10 (P < 0.05), and due to significantly more LDs per cell between days ∼10 and ∼17 (P < 0.05). The statically stretched cells also differentiated significantly faster than the nonstretched cells within the first ∼10 days (P < 0.05). Adding peroxisome proliferator-activated receptor-γ (PPARγ) antagonist did not change these trends, as the %-lipid area per FOV in the stretched cultures that received this treatment was still significantly greater than in the nonstretched cultures without the PPARγ antagonist (14.44 ± 1.96% vs. 10.21 ± 3%; P < 0.05). Hence, the accelerated adipogenesis in the stretched cultures was not mediated through PPARγ. Nonetheless, inhibiting the MEK/MAPK signaling pathway reduced the extent of adipogenesis in the stretched cultures (13.53 ± 5.63%), bringing it to the baseline level of the nonstretched cultures without the MEK inhibitor (10.21 ± 3.07%). Our results hence demonstrate that differentiation of adipocytes can be enhanced by sustained stretching, which activates the MEK signaling pathway.

A method for quick, low-cost automated confluency measurements.

A culture's confluency is a fundamental measure in the field of biology, and routine quantification of confluence in cell culture protocols, biological assays and tissue engineering work is important. However, current techniques for obtaining confluency are either subjective, destructive, not simple enough, or time-consuming. We developed an image processing method for automated confluency measurement from a single microscope image without any chemical staining. To demonstrate utility we monitored the confluency of three cell types: NIH3T3 fibroblasts, C2C12 myoblasts, and 3T3L1 pre-adipocytes for 5 days, twice a day. The captured micrographs had different and uneven illumination, the cell types varied in cell-to-background contrast, and the confluency ranged between 10% and 100%. Despite these variable conditions, our method was shown to be practical, economic, and easy to implement, providing quantitative confluency measurements over time in each culture case. The method is hence suitable for routine automatic determination of confluency to standardize handling of cells, achieve reproducibility across trials, and improve accuracy in experimental outcome measures.

Seaweed cellulose scaffolds derived from green macroalgae for tissue engineering.

Extracellular matrix (ECM) provides structural support for cell growth, attachments and proliferation, which greatly impact cell fate. Marine macroalgae species Ulva sp. and Cladophora sp. were selected for their structural variations, porous and fibrous respectively, and evaluated as alternative ECM candidates. Decellularization-recellularization approach was used to fabricate seaweed cellulose-based scaffolds for in-vitro mammalian cell growth. Both scaffolds were confirmed nontoxic to fibroblasts, indicated by high viability for up to 40 days in culture. Each seaweed cellulose structure demonstrated distinct impact on cell behavior and proliferation rates. The Cladophora sp. scaffold promoted elongated cells spreading along its fibers' axis, and a gradual linear cell growth, while the Ulva sp. porous surface, facilitated rapid cell growth in all directions, reaching saturation at week 3. As such, seaweed-cellulose is an environmentally, biocompatible novel biomaterial, with structural variations that hold a great potential for diverse biomedical applications, while promoting aquaculture and ecological agenda.

The effects of external counter pulsation therapy on circulating endothelial progenitor cells in patients with angina pectoris.

OBJECTIVES: External counter pulsation therapy (ECPT) offers symptomatic relief and improves ischemia in patients with refractory angina pectoris. We aimed to determine the effects of ECPT on circulating endothelial progenitor cells (EPCs). METHODS: We prospectively studied 25 patients with angina pectoris treated with ECPT (n = 15) or receiving standard care (n = 10). The number of EPCs positive for CD34 and kinase insert domain receptor (KDR) was determined by flow cytometry and the number of colony-forming units (CFUs) was assessed in a 7-day culture, before ECPT and after 9 weeks. RESULTS: ECPT improved anginal score from a median of 3.0 to 2.0 (p < 0.001). Concomitantly, ECPT increased EPC number from a median of 10.2 to 17.8/10(5) mononuclear cells (p < 0.05), and CFUs from 3.5 to 11.0 (p = 0.01). Flow-mediated dilatation was improved by ECPT from 7.4 to 12.2% (p < 0.001) and correlated with EPC-CFUs (r = 0.461, p = 0.027). The levels of asymmetric dimethylarginine were reduced by ECPT from 0.70 to 0.60 micromol/l (p < 0.01). In contrast, the same parameters did not change in the control group, before and after follow-up. CONCLUSIONS: The present pilot study shows, for the first time, that ECPT is associated with increased number and colony-forming capacity of circulating EPCs.

Low-intensity ultrasound induces angiogenesis in rat hind-limb ischemia.

We investigated the effect of low-intensity ultrasound (US) on tissue blood flow and angiogenesis after limb ischemia in vivo. Rats underwent surgical ligation of the femoral or the iliac arteries. Half the animals were exposed to low-intensity US (0.05 W/cm2) during three consecutive sessions. At 3 weeks postsurgery, limb perfusion was assessed using laser Doppler and angiography. Immunostaining and vascular endothelial growth factor (VEGF) messenger ribonucleic acid (mRNA) expression were performed 7 d postsurgery. US irradiation significantly improved limb perfusion in both ischemic models (p = 0.04). Angiography showed increased blood vessels in the moderate ischemia (p = 0.01), but not in the severe ischemia (p = 0.19). Histology demonstrated a significantly higher number of blood vessels and proliferating cells in US-irradiated moderate and severe ischemia (p = 0.002 and p = 0.03, respectively). VEGF mRNA was significantly higher in moderate ischemia (p = 0.02). No differences in apoptotic cell death were evident in the models. Low-intensity US significantly improved tissue blood flow and angiogenesis, irrespective of the extent of the ischemia. (E-mail: ).

A phase-contrast microscopy-based method for modeling the mechanical behavior of mesenchymal stem cells.

We present three-dimensional (3D) finite element (FE) models of single, mesenchymal stem cells (MSCs), generated from images obtained by optical phase-contrast microscopy and used to quantify the structural responses of the studied cells to externally applied mechanical loads. Mechanical loading has been shown to affect cell morphology and structure, phenotype, motility and other biological functions. Cells experience mechanical loads naturally, yet under prolonged or sizable loading, damage and cell death may occur, which motivates research regarding the structural behavior of loaded cells. For example, near the weight-bearing boney prominences of the buttocks of immobile persons, tissues may become highly loaded, eventually leading to massive cell death that manifests as pressure ulcers. Cell-specific computational models have previously been developed by our group, allowing simulations of cell deformations under compressive or stretching loads. These models were obtained by reconstructing specific cell structures from series of 2D fluorescence, confocal image-slices, requiring cell-specific fluorescent-staining protocols and costly (confocal) microscopy equipment. Alternative modeling approaches represent cells simply as half-spheres or half-ellipsoids (i.e. idealized geometries), which neglects the curvature details of the cell surfaces associated with changes in concentrations of strains and stresses. Thus, we introduce here for the first time an optical image-based FE modeling, where loads are simulated on reconstructed 3D geometrical cell models from a single 2D, phase-contrast image. Our novel modeling method eliminates the need for confocal imaging and fluorescent staining preparations (both expensive), and makes cell-specific FE modeling affordable and accessible to the biomechanics community. We demonstrate the utility of this cost-effective modeling method by performing simulations of compression of MSCs embedded in a gel.

Thyroid hormone up-regulates Na+/K+ pump alpha2 mRNA but not alpha2 protein isoform in cultured skeletal muscle.

Thyroid hormone (T(3)) is known to up-regulate the physiological expression of the Na(+)/K(+) pump in cultured skeletal muscle. We recently reported that primary cultured rat skeletal muscle expresses only the alpha(1), beta(1) and beta(2) protein isoforms of Na(+)/K(+) pump. Interestingly, alpha(2) mRNA is detectable while the alpha(2) protein isoform is not. We therefore examined whether T(3) might up-regulate the expression of Na(+)/K(+) pump alpha(2) isoform at the protein and mRNA level. We also examined the regulation by this hormone of the other isoforms of the pump. Primary cultures were treated with T3 for 48 h from day 4 to day 6 of differentiation. Protein and mRNA isoforms of Na(+)/K(+) pump were identified by Western blotting and Northern blotting, respectively. T(3) induced a marked increase in the beta(1) protein and a slight increase in the alpha(1) protein. T(3) did not affect expression of the beta(2) protein. The alpha(2) protein was not detected in either untreated or T(3)-treated cells. In contrast, alpha(2) mRNA was highly up-regulated by T(3) treatment compared to the other isoforms. The lack of expression of the alpha(2) protein isoform following T(3) treatment suggests that posttranscriptional events related to this isoform may be dependent on other growth factors or hormones.

A new technique for studying directional cell migration in a hydrogel-based three-dimensional matrix for tissue engineering model systems.

Cell migration has a key role in biological processes, e.g. malignancy, wound healing, immune response and morphogenesis. Studying migration and factors that influence it is beneficial, e.g. for developing drugs to suppress metastasis, heal wounds faster or enhance the response to infection. Though the majority of the literature describes two-dimensional (2D) migration studies in culture dishes, a more realistic approach is to study migration in three-dimensional (3D) constructs. However, simple-to-implement, straight-forward standardized quantitative techniques for analysis of migration rates of cell colonies in 3D are still required in the field. Here, we describe a new model system for quantifying directional migration of colonies in a hyaluronic acid (oxi-HA) and adipic acid dihydrazide (ADH) hydrogel-based 3D matrix. We further demonstrate that our previously reported image processing technique for measuring migration in 2D (Topman et al., 2011, 2012) is extendable for analyzing the rates of migration of cells that directionally migrate in the hydrogel and are fluorescently stained with a 4',6-diamidino-2-phenylindole (DAPI) nuclear stain. Together, the present experimental setup and image processing algorithm provide a standard test bench for measuring migration rates in a fully automated, robust assay which is useful for high-throughput screening in large-scale drug evaluations, where effects on migration in a 3D matrix are sought.

A standardized objective method for continuously measuring the kinematics of cultures covering a mechanically damaged site.

The kinematics of cell migration is frequently being studied in the context of wound healing. Scratch wound assays in vitro are particularly popular, being a cost-effective method for characterizing the kinematic parameters of cultures. However, currently there are no objective and standardized measures of these kinematic parameters. We addressed these issues by developing an automatic and quantitative method for determining time-dependent damage areas in "wound healing" monolayer culture experiments by means of image processing. "Wound" area over time data are then fitted to a Richards function (non-symmetric sigmoid) from which we determine the migration rate, time for onset of mass cell migration and time for end of mass cell migration. We demonstrate the utility of our present method by conducting "wound healing" experiments in 8 cultures of NIH3T3 fibroblast cells which were monitored by time-lapse microscopy. The measures derived from Richards function fits to the area-time curves (normalized root mean squared errors ≤3.8%) are calculated based on the entire time course of the data. Accordingly this method is substantially more reliable than the common practice where migration rates are determined based on two time points (start and end stages of migration).

Transplantation of genetically engineered cardiac fibroblasts producing recombinant human erythropoietin to repair the infarcted myocardium.

BACKGROUND: Erythropoietin possesses cellular protection properties. The aim of the present study was to test the hypothesis that in situ expression of recombinant human erythropoietin (rhEPO) would improve tissue repair in rat after myocardial infarction (MI). METHODS AND RESULTS: RhEPO-producing cardiac fibroblasts were generated ex vivo by transduction with retroviral vector. The anti-apoptotic effect of rhEPO-producing fibroblasts was evaluated by co-culture with rat neonatal cardiomyocytes exposed to H2O2-induced oxidative stress. Annexin V/PI assay and DAPI staining showed that compared with control, rhEPO forced expression markedly attenuated apoptosis and improved survival of cultured cardiomyocytes. To test the effect of rhEPO on the infarcted myocardium, Sprague-Dawley rats were subjected to permanent coronary artery occlusion, and rhEPO-producing fibroblasts, non-transduced fibroblasts, or saline, were injected into the scar tissue seven days after infarction. One month later, immunostaining identified rhEPO expression in the implanted engineered cells but not in controls. Compared with non-transduced fibroblasts or saline injection, implanted rhEPO-producing fibroblasts promoted vascularization in the scar, and prevented cell apoptosis. By two-dimensional echocardiography and postmortem morphometry, transplanted EPO-engineered fibroblasts did not prevent left ventricular (LV) dysfunction and adverse LV remodeling 5 and 9 weeks after MI. CONCLUSION: In situ expression of rhEPO enhances vascularization and reduces cell apoptosis in the infarcted myocardium. However, local EPO therapy is insufficient for functional improvement after MI in rat.