Fibrillin-1 and -2 differentially modulate endogenous TGF-β and BMP bioavailability during bone formation

Harikiran Nistala, Sui Lee-Arteaga, Silvia Smaldone, Gabriella Siciliano, Luca Carta, Robert N. Ono, Gerhard Sengle, Emilio Arteaga-Solis, Regis Levasseur, Patricia Ducy, Lynn Y. Sakai, Gerard Karsenty, and Francesco Ramirez

ABSTRACT

Extracellular regulation of signaling by transforming growth factor (TGF)–β family members is emerging as a key aspect of organ formation and tissue remodeling. In this study, we demonstrate that fibrillin-1 and -2, the structural components of extracellular microfibrils, differentially regulate TGF-β and bone morphogenetic protein (BMP) bioavailability in bone. Fibrillin-2–null (Fbn2-/-) mice display a low bone mass phenotype that is associated with reduced bone formation in vivo and impaired osteoblast maturation in vitro. This Fbn2-/- phenotype is accounted for by improper activation of latent TGF-β that selectively blunts expression of osterix, the transcriptional regulator of osteoblast maturation, and collagen I, the structural template for bone mineralization. Cultured osteoblasts from Fbn1-/- mice exhibit improper latent TGF-β activation as well, but mature faster because of increased availability of otherwise matrix-bound BMPs. Additional in vitro evidence excludes a direct role of microfibrils in supporting mineral deposition. Together, these findings identify the extracellular microfibrils as critical regulators of bone formation through the modulation of endogenous TGF-β and BMP signaling.

SUMMARY

INTRODUCTION

The extra-cellular matrix (ECM) has been shown to be an important component of growth signaling and development. Amongst the ECM proteins, fibrillins and their associated proteins have been found to be important for cell maturation and growth and are typically bound by TGF-β and BMP. This paper looks at the effects of mutated proteins/genes for fibrillin-1 and -2 individually, focusing on TGF-β and BMP and their effects on osteoblast growth in the context of Marfan Syndrom (MFS) and congenital contractural arachnodactyly (CCA).

Figure 9. Model of microfibril-mediated control of osteoblast maturation. The scheme summarizes the distinct contributions of osteoblast-produced fibrillin-1 and fibrillin-2 microfibrils to osteogenic differentiation through the differential regulation of endogenous TGF-β and BMP signals that together calibrate the rate of bone formation.

RESULTS

Figure 1. Reduced bone mass and BFR in Fbn2-/- mice. (A) Representative von Kossa staining and μCT images of vertebral sections from 3-mo-old WT and Fbn2-/- male mice with histograms summarizing the μCT measurements of volumetric bone mineral density (BMD) and BV/TV in these samples. (B) Illustrative examples of dual-calcein labeling in tibiae of 3-mo-old WT and Fbn2-/- male mice with histograms summarizing BFR values and osteoblast numbers in WT and mutant samples. Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P < src="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEgPj1kD5z84xd0aK2J6vx-AQxnycSmFfx0oE1m2GRTo2m9dBwBUxFs-AvCrlTY-NNjG9Z5aDhLEze54jSiYMlnqNMofWhxw8W-0YN1QrlsvkFIBqWUbh5LrsEEm5AWbYa2zPKcn/s200/fig2.bmp" style="display: block; margin: 0px auto 10px; text-align: center; cursor: pointer; width: 178px; height: 200px;" alt="" id="BLOGGER_PHOTO_ID_5534865547216898946" border="0">

Figure 2. Impaired maturation of Fbn2-null osteoblasts. (A) Illustrative images of von Kossa staining of neonatal cOb (left) and adult MSCs (right) along with magnified van Geison–counterstained images (middle) of cOb differentiated for 21 d after OS administration. Histograms summarize the number of mineralized nodules in WT and Fbn2-null (Fbn2-/-) cOb (n = 15) and MSC (n = 5) cultures. Bars, 200 μm. (B) AP activity of WT and Fbn2-/- cOb measured 3 d before (day -3) and 4 d after (day 4) OS administration (day 0) and normalized to total protein levels (n = 5). (C) qPCR estimates of indicated transcripts in total RNA isolated from day 4 cOb cultures (n = 4; top) or P4 calvariae of WT and Fbn2-/- mice (n = 4; bottom). (D and E) MTT and BrdU incorporation assays (D) and C-myc and Ccnd1 mRNA levels (E) at the indicated days of cOb differentiation (n = 6). (F and G) Cell survival evaluated by Trypan blue exclusion (F; n = 4) and cell apoptosis (G) measured as the fraction of cleaved caspase-3 over full-length protein (with histograms representing densitometric analyses) in WT and mutant cObs. Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P <>

Figure 3. Osterix and collagen down-regulation in Fbn2-null osteoblasts. (A and C) Illustrative images of pOBCol2.3GFP (A) or Osx-GFP::Cre transgene expression (C) in Fbn2-null and WT cOb at day 17 or 4 of differentiation, respectively, with histograms summarizing the number of GFP-positive cells (n = 3); nuclei are DAPI stained. (B) Illustrative images of tibiae of Fbn2-/- and WT mice harboring the pOBCol2.3GFP transgene that include (from top to bottom) hematoxylin/eosin staining, GFP expression, and magnified views of toluidine- and GFP-positive cells; histograms summarize cell counts in the last images (n = 3). (D) von Kossa staining after 21 d of differentiation of cOb treated with or without 100 ng/ml rhBMP2 with histograms (right) summarizing numbers of mineralized nodules and levels of Osx and Col1a2 transcripts in mutant and WT samples (n = 3). Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P <>

Figure 4. Fibrillin-2 controls TGF-β bioavailability. (A) Surface plasmon resonance sensograms of binding of immobilized L1K (left) and L4K (right) to rF86 at concentrations between 0 (baseline tracing) and 200 nM (top tracing). Binding was recorded as resonance units (RU), and nonspecific binding to control surface was subtracted at a molar ratio of 1:1. The table summarizes the affinity data (expressed as Kd) for interactions between the LTBPs and fibrillin-2 peptide. (B) Immunodetection of nuclear pSmad2 accumulation in day 4 cOb cultured in low serum with or without SB431542; nuclei are DAPI stained. Histograms summarize the percentage of pSmad2-positive nuclei in WT (black) and mutant (gray) cells (n = 3). (C) Transcriptional activity of p3TP-lux reporter plasmid transfected in WT or Fbn2-null cOb cultured in low serum (n = 3). (D) TMLC bioassays (n = 5) measuring active TGF-β in WT or Fbn2-null cOb cultures (left) or total TGF-β in heat-activated conditioned media of the same cultures (right). (E) qPCR estimates of Tgf-β transcripts in WT and Fbn2-null cOb (n = 3). Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P <>

Figure 5. Elevated TGF-β signaling limits Fbn2-null osteoblast maturation. (A) Illustrative images of immunoreactive material corresponding to the indicated proteins deposited in the ECM of overconfluent WT, Fbn1-null, and Fbn2-null cOb cultures after 4 d of differentiation; nuclei are DAPI stained. Bars, 50 μm. (B) Maturation of WT and Fbn2-null cOb cultures treated with 1 μM SB431542, 300 ng/ml neutralizing pan–TGF-β antibody, or 50 μM Alk5 siRNA with histograms summarizing the number of mineralized nodules in each treatment (n = 3). (C) qPCR estimates of the indicated mRNA levels in the Alk5 silencing experiments. Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P <>

Figure 6. Abnormally high TGF-β activity in differentiating Fbn1-null osteoblasts. (A) Illustrative images of von Kossa–stained WT and Fbn1-null (Fbn1-/-) cOb after 21 d of differentiation with histograms summarizing the number of mineralized nodules in each sample (n = 5). (B) Cell proliferation of WT and mutant cOb at day -3 of cell culture as assessed by BrdU incorporation and qPCR quantification of C-myc and Ccnd1 transcripts (n = 3). (C and D) qPCR estimates of indicated transcripts in total RNA isolated from day 4 differentiating WT and mutant cOb cultures (C; n = 4) and from P4 WT and Fbn1-/- calvarial bones (D; n = 3). (E) TMLC bioassays (n = 5) measuring active TGF-β in WT or Fbn1-null cOb cultures (left) or total TGF-β in heat-activated conditioned media of the same cultures (right). (F) Transcriptional activity of p3TP-lux reporter plasmid transfected in WT or Fbn1-null cOb cultured in low serum with or without 1 μg/ml of noggin (n = 3). (G) qPCR estimates of TGF-β transcripts in WT and Fbn2-null cOb (n = 3). Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P <>

Figure 7. Fibrillin-1 regulates BMP signaling in cultured osteoblasts. (A) Immunodetection of nuclear pSmad1/5/8 accumulation in day 4 differentiating WT and Fbn1-null (Fbn1-/-) and Fbn2-null (Fbn2-/-) cOb cultured in low serum; nuclei are DAPI stained, and histograms summarize the percentage of pSmad1/5/8-positive nuclei in WT (black), Fbn1-null (white), or Fbn2-null (gray) cells (n = 3). Bars, 25 μm. (B) Transcriptional activity of pBRE-lux reporter plasmid in day 4 differentiating WT, Fbn1-null, and Fbn2-null cOb (n = 5). (C) C2Cl2BRA bioassay measuring BMP signaling in conditioned media from WT, Fbn1-null, and Fbn2-null cOb and from the first two cell cultures treated with either 1 μg/ml noggin or 1 μM SB431542 (n = 3). (D and E) qPCR estimates of Bmp transcripts in day 4 Fbn1-/- and Fbn2-/- cOb cultures, respectively (n = 3). Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P < id="p-3">

Figure 8. Microfibrils are not a structural substrate for matrix mineralization. (A) Illustrative von Kossa–stained WT cOb cultures in which Fbn1 or Fbn2 expression was silenced by RNAi with histograms on the immediate and far right showing the number of mineralized nodules and levels of indicated transcripts (n = 3), respectively. (B) Illustrative von Kossa–stained Fbn2-/- cOb cultures in which Fbn1 was silenced with histograms on the immediate and far right, showing the number of mineralized nodules and levels of indicated transcripts, respectively (n = 3). Error bars indicate mean ± SD, and asterisks indicate statistically significant differences (P < href="https://blogger.googleusercontent.com/img/b/R29vZ2xl/AVvXsEjDeAk42Bq4q3eP7jMCQcWz9gL_SOkNd5F9Eh800aXmrwE5oFq1TxpUGjF1w-qC8uEf9hC7aIaaTo1y2EIT_HN_rjSIoPKhUaX4_yY-4IX49neG3JiBi60SY7U0hgobqKvAN4Eq/s1600/fig9.bmp">

DISCUSSION

In short, fibrillin-1 which is associated with MFS is shown to impair bone growth, as the absence of the related gene results in faster maturation than typical cells. On the other hand, the lack of fibrillin-2 is associated with CCA results in impaired "bone formation and osteoblast maturation." In the end, it can be said that the combination of fibrillins as well as their associated and binding proteins are responsible for both "anabolic and catabolic phases of bone homeostasis."

The TGF-β and BMP proteins are important in the specificity of the cellular signaling and resulting cascades. The combination of these two proteins is important only in the combined number of the two. The relative amounts in that sum has been shown to be unimportant. All of these effects, however, are said to be bone-specific after observing the effects of MFS and CCA. The details of this specificity is still unknown and needs to be further explored for a more comprehensive understanding of these protein effects and diseases.

METHODS

Experiments were done on both mice and cultured cells, ranging from no manipulation to absences of either the fibrillin-1 or -2 genes. The cultured cells were primary cultures of osteoblasts and mesenchymal stem cells (MSCs). RNA was collected with RNeasy Mini kits, and results were analyzed using immunostaining, qPCR, and a variety of bioassays (i.e. Surface Plasmon Resonance - SPR).

Critique

The conclusions drawn from the data of cultured cells were for endogenous activity; however, the experiments used exogenous stimulators/inhibitors. These in vitro conclusions are probably reasonably reliable however, as it is probably the activity levels rather than the activators themselves that matter. It would still have been nice if there was some way to implement an endogenous set of stimulators/inhibitors, so that the conclusions could be definite. On the other hand, the experiments on the modified mice did prove to be amongst the first to unambiguously show the importance of the fibrillin bindings and associations in the ECM for osteogenic differentiation. The combination of the findings from the cultured cells and modified mice also serve to reinforce the conclusions drawn from the in vitro experiments.

A couple of issues were not dealt with in these set of experiments. Firstly, the other cascades that could have resulted from high TGF-β activity were not accounted for. Any other resulting cascade could have influenced the observed effects or even produced some nuances. There is no reason to state that the observations and results are the effect of this single pathway. Another issue left untouched was the interactions between signaling events. Events occur in both the space and time dimensions and the signals are bound to have some sort of effect on each other. Spatiotemporal signaling events and consequences were not accounted for, and this something that should be looked into.

Increased phosphotyrosine content and inhibition of proliferation in EGF-treated A431 cells

http://www.nature.com/nature/journal/v293/n5830/pdf/293305a0.pdf

Nature 293, 305 - 307 (24 September 1981); doi:10.1038/293305a0

Gordon N. Gill & Cheri S. Lazar
Department of Medicine, Division of Endocrinology, University of California, San Diego, School of Medicine, La Jolla, California 92093, USA

Abstract

Epidermal growth factor (EGF), which binds to specific high-affinity cell-surface receptors, stimulates replication of a number of cell types1,2. In vitro EGF stimulates a membrane-associated protein kinase which catalyses phosphorylation of the EGF receptor at tyrosine residues3,4. The transforming proteins of several RNA tumour viruses are protein kinases which also specifically catalyse phosphorylation at tyrosine residues5−10. An elevated level of phosphotyrosine is found in cells transformed by Rous sarcoma, Fujinami sarcoma, PRCII, Y73, Snyder−Theilin and Gardner−Armstrong feline sarcoma and Abelson murine leukaemia viruses5,10−12. At least four of these viruses, which encode distinct protein kinases, catalyse phosphorylation of tyrosine residues in the same cellular substrate proteins13. In vitro EGF-stimulated protein kinase catalyses the phosphorylation of anti-p60src heavy chains, suggesting that this enzyme recognizes similar substrate determinants to p60src (refs 14,15). Here we demonstrate that EGF treatment of A431 human epidermoid carcinoma cells increases phosphotyrosine content, indicating that EGF stimulates tyro sine-specific protein kinase activity in vivo as well as in vitro. In contrast to Rous sarcoma virus (RSV) transformation, EGF inhibits replication of A431 cells. This inhibition by EGF is influenced by both cell density and tissue culture substratum.

Summary

A431 human epidermoid carcinoma cells have an unusually high density of EGF receptors. It has been known that EGF increases the phosphotyrosine content of exponentially growing A431 cells. EGF stimulates a membrane-associated protein kinase through phosphorylation. EGF increased the phosphotyrosine content of A431 cells sevenfold (0.14 and 0.02% in EGF-treated and untreated A431 cells respectively), indicating that EGF activates tyrosine-specific protein kinase activity in vivo.



Figure.1
Exponentially growing A431 cells were cultured. In control, no EGF was given while in EGF group, EGF was added. After culturing, cells were broken down and the proteins were collected. Electrophoresis was done and following Western blot was done and then overexposed to phosphoamino acids. The results shows phosphorylated tyrosine(p-tyr) only exists in EGF-treated cells.


Figure 2.
Effect of EGF on growth of A431 cells plated at different cell densities. Cells were cultured with different densities: 550(□), 2500(○) and 11000(●) cells / cm^2. The possibility that increased EGF degradation at high cell densities on tissue culture dishes contributed to the rightward shift in the dose-response curve shown in Figure 2.


Figure 3.
Effect of cell density on binding of EGF to A431 cells. A431 cells were subcultured into 3-cm tissue culture dishes and used 24-48 hr later. EGF-binding to A431 cells grown on tissue culture dishes: ●, I-EGF binding of constant specific activity. KD is the equilibrium dissociation constant. This figure shows that there is an inverse relationship between cell density and the affinity of EGF receptors for EGF.


Critique

Epithermal growth factor(EGF) is one of the molecules that cancer researchers are interested in. Many cancer cells have extraordinarily high density of EGF-receptors, and EGF is known to affect cell proliferation and apoptosis by phosphorylating EGF receptors.

This article is related to our groups project, whis is to see the effect of EGF on A431 cells in their morphology and proliferation. This research done by Gill and Lazar is one of the beginning researches of EGF on cancer cells. At the time of this research, the molecular activity of EGF on EGF receptor was figured out: in vitro, EGF was found out that it catalysed the phosphorylation of the EGF receptor at tyrosine residues. The main purpose of this research was to see the effect of EGF in vivo.

This article was published on Nature in 1981, so it does not show the current hot issues in molecular biology/bioengineering. However, it shows several interesting points that are worth taking a look. First, this research began as to see the effect of EGF in vivo, but as the experiment went on, the authors could discover a new experimental result that the effect of EGF was influenced by both cell density and tissue culture environment. This shows that even we start an experiment with a clear hypothesis and purpose, we may make unexpected but new discoveries.

Secondly, the methods used in this research are outdated and different from current methods. For example, if you look at Figure 1 which shows the protein electrophoresis result, you will easily see that the figure seems messy. The purpose of the electrophoresis was to visualize the amount of phosphorylated tyrosine in EGF-treated cells and control, so the dark background is actually a noise. Considering the fact the phosphorylated tyrosine had been studied well in vitro before this research was performed, I think there was a reason that they could not perform immunoblotting skills, which would have clearly shown the difference in amount of p-tyrosine. In my opinion, Western blot was not popular at the time of this research, because the Western blot was only invented in 1975, a few years before this research. Nevertheless, the researchers proved that there was significant difference in the amount of p-tyrosine by manually separating the proteins.

Lastly, this article does not have small titles as "introduction", "materials & methods", and "results" which can be found in most of today's articles. By reading an article like this, I realized that those small titles expedite the understanding of readers.

To sum up, this article is interesting because it is one of the first researches to see the effect of EGF in vivo. Even though it is from 1981, the skills and logic through the experiment are understandable, and it will let you have an opportunity to think about current research techniques in different way.

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity

Tsai, J., Lee, J.T., Wang, W. et al. (2008). Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc. Natl. Acad. Sci. U.S.A. 105, 3041–3046.

Background

Mutations in the BRAF gene occur in a wide variety of human tumors. Specifically, the BRAF V600E missense mutation is the most frequent oncogenic protein kinase mutation known. Tumor phenotypes correlated with oncogenic BRAF mutations include increased severity of cancer and decreased response to chemotherapy. One therapeutic approach to treating BRAF mutant cancer involves direct inhibition of the oncogenic BRAF kinase activity. Thus, in BRAF V600E mutants, targeted inhibition of the BRAF V600E gene product may be an important avenue to treating such tumors, especially in traditional therapy-resistant tumor types. The development of highly specific and effective inhibitors of the BRAF V600E gene product would provide insight into the therapeutic relevance of this target in malignant tumors. In this paper, the authors did exactly such by discovering the BRAF V600E specific inhibitor PLX4720 and examining its efficacy in both cell and animal based models.

Scaffold- and Structure-Based Discovery of the Inhibitor

The paper started off with a discussion of the rational drug design process that led to the discovery of PLX4720. Scaffold and structure based discovery was utilized. In other words, protein kinase scaffolds were first identified for a selected library of 20,000 compounds. The compounds were applied at 200 µM to multiple solved, but structurally divergent protein kinases. The compound-kinase structures were then screened by co-crystallography to identify which compounds would best bind to the known kinase scaffolds. A tremendous amount of work went into such screening process as over 100 structures showing bound compound were solved in this manner. Once productive binding activity was discovered for a given compound, chemical analysis was then performed to modify the compound for further inhibitory binding to the protein kinase. Through such a characterization process, PLX4720 was discovered to selectively bind to the BRAF V600E gene product (See Figure 1 for the PLX4720 binding structure and other structures leading up to its discovery). In fact, the authors claim, based on IC50 analysis (that is, the concentration at which 50% of the kinase is inhibited) PLX4720 binds with greater than 10-fold selectivity for BRAF V600E than for the wild-type BRAF.

Fig. 1. Structures of individual compounds leading to the discovery of PLX4720 are shown. (A) The chemical structure of 3-aminophenyl-7-azaindole (compound 1) is shown beneath its costructure with Pim-1 kinase. (B) The chemical structure of 3-(3-methoxybenzyl)-7-azaindole (compound 2) is shown beneath its costructure with the kinase domain of FGFR1. (C) The chemical structure of PLX4720 is shown beneath its costructure with B-Raf kinase.

Modes of Inhibitor Interaction and Selectivity

After characterizing the discovery process, the authors move on to further describe the binding action between PLX4720 and BRAF V600E. Crystallographic analysis revealed that there may be multiple conformations to the structure’s bound state. Some of these conformations reflect the “active” state of PLX4720, that is, the state in which the compound inhibits BRAF V600E, while other conformations reflect an “inactive” state. Such conformations and their respective binding interactions are displayed in Figure 2.

Fig. 2. Depiction of the three-dimensional structure of PLX4720 bound to B-Raf. (A) The structure of B-Raf^V600E bound to PLX4720 (yellow) is overlayed with an ATP model based on structures of ATP analogs in complex with other tyrosine kinases (orange). This view indicates that the PLX4720 scaffold overlaps with the adenine-binding site, but the tail of PLX4720 binds to a different pocket from the ATP ribose-triphosphate tail. The positions of the hinge, activation loop (A-loop), and phosphate-binding loop (P-loop) are also shown. (B) A surface representation shows PLX4720 binding to the B-Raf-selective pocket in the active conformation. (C) A surface representation shows PLX4720 binding to the kinase general pocket in the inactive conformation. (D) A close-up view shows the overlay PLX4720 bound to both active (green) and inactive (purple) conformations of the V600 protein, and PLX3203 (yellow) bound to V600E protein in the active kinase conformation. (E) A stereoview shows the specific interactions of PLX4720 to the active kinase conformation. In this conformation, the phenylalanine of the DFG loop is pointing in toward the compound-binding site. (F) A stereoview shows the specific interactions of PLX4720 to the inactive kinase conformation. In this conformation, the phenylalanine of the DFG loop is pointing away from the compound-binding site, and binding of PLX4720 is disfavored, leading to partial occupancy of this site even at the 1 mM compound concentration used in cocrystallography.

Cellular Selectivity in Multiple Tumor Lines

PLX4720 was then tested for its selectivity for BRAF V600E in multiple mutant and wild type cell lines. Each cell line was incubated for 1 hour in various concentrations of PLX4720 treatment. Two outputs were then measured, the GI50 (50% of growth inhibition) of cellular proliferation and GI50 of P-ERK expression. A previously characterized MEK inhibitor, PD0325901, was used as a control to benchmark P-ERK expression levels. It was also shown that PLX4720 is even more selective for BRAF V600E in cell lines than in kinase alone reactions. In some cases, the selectivity was up to over 100 fold. In addition, the clear down-regulation of P-ERK indicated that PLX4720 critically affected BRAF-MEK-ERK pathway. Notably, the BRAF-MEK-ERK pathway is generally regulated by feedback mechanisms in wide-type tumor cell lines, that is, even with BRAF inhibition, P-ERK is not critically down-regulated. Interestingly, this is not the case in BRAF V600E mutants, as demonstrated by the critical down regulation of P-ERK. The authors speculate that the negative feedback loop is lost in BRAF V600E cells, resulting in a pronounced dependence on the BRAF-MEK-ERK pathway.

Melanoma cell biology

To further drive the point home, the authors then demonstrated the effect of PLX4720 inhibition on a larger panel of melanoma cell lines. P-ERK expression was examined by Western blotting, and representative blots were shown in Figure 3A. Of the cell lines tested, 1204Lu (a BRAF V600E line) and C8161 (a BRAF wild-type line) were selected for further study because these cell lines come from highly malignant tumors that were resistant to chemotherapy. After administering varying concentrations of PLX4720 to these cell lines, it was demonstrated that 1204Lu displayed lower cell viability, while C8161 was unaffected (Figure 3B). This indicated that PLX4720 may have potential clinical applications where traditional chemotherapy has failed. In Figures 3C and 3D, an Annexin V PI stain coupled with cell flow cytometry was utilized to characterize 1205Lu and C8161 when treated with different concentrations of PLX or with the same concentration of PLX at different time points. The Annexin stains for alive vs. apoptotic cells, while the propidium iodide (PI) stains for necrotic vs. non-necrotic apoptotic cells. The results indicate that under equal PLX4720 treatment conditions, more cells in the V600E line underwent apoptosis than cells in the wide-type line. Again, this is consistent with the authors’ claims that PLX4720 selectively inhibits BRAF V600E. In Figure 3E, a live dead assay using Calcein-AM and EtBr was applied under varying treatment concentrations of PLX4720. Once again, it is shown that the BRAF V600E mutant line was more critically affected by PLX4720 and had more dead cells than the wild-type line. In Figure 3F and 3G, synthetic skin was created using 1204Lu (BRAF V600e) and C8161(BRAF wild-type) lines. The synthetic skin were respectively treated with PLX and immunostained with DAPI (for DNA), S100 (indication of cell proliferation), PCNA (indication of cell proliferation), and TUNEL (for DNA fragmentation). In all stains indicating cell growth and viability, it is shown that there is weaker signal in the PLX4720 treated BRAF V600E mutant synthetic skin, while DNA fragmentation is shown to be increased under PLX4720 treatment. Meanwhile, all 5 stains appear to be relatively unchanged after PLX treatment in the BRAF wild-type synthetic skin. This again served to drive the nail in the coffin in saying that PLX4720 selectively inhibits BRAF V600E lines.

Fig. 3.

Selectivity and antimelanoma activity of PLX4720 in vitro. (A) Panel of melanoma cell lines (V600E+, left; B-Raf wild-type, right) were treated with various dosages of PLX4720, and protein extracts were subject to immunoblotting. Activity within the MAPK pathway is represented by levels of phosphorylated ERK; β-actin serves as a loading control. (B) B-Raf V600E+ (Left) and B-Raf wild-type (Right) cells were treated PLX4720 at the indicated dosages for 72 h. Cell number was assayed by MTT analysis. (C) 1205Lu and C8161 cells were treated with 1 μM PLX4720 for the times indicated and stained with Annexin V/FITC and propidium iodide (PI) for analysis of apoptosis. (D) Graphs represent raw data from the Annexin/PI assay. (E) Spheroids from 1205Lu and C8161 cells were treated with indicated dosages of PLX4720 and stained with calcein AM and ethidium bromide to assess overall viability. Green (calcein-AM) indicates live cells; red (EtBr) depicts apoptotic cells. (F) Synthetic skin was created by using 1205Lu (V600E+) cells and subjected to vehicle control (Upper) or 1 μM PLX4720 (Lower) for 72 h. H&E staining is depicted (Left), and immunofluorescent stains for DAPI, PCNA, and S100 are also shown. (G) Same as F, except with C8161 (B-Raf wild-type) cells.

Animal Efficacy

In addition to in vitro study, PLX4720 was further tested in mouse models for its efficacy to cause tumor regressions in vivo. A tumor xenograft of COLO205 cells (which are BRAF V600E mutant) was planted into nude mice. The xenograft mice then treated with either vehicle control, 5 mg/kg PLX4720, or 20 mg/kg PLX4720 by oral gavage daily on days 1-14 of the 25 day study. The results (Figure 4A) indicate that with increasing PLX4720 dosage, there was less tumor growth in the mice. In their style of driving the point home repeatedly, the authors then again performed xenografts using 1205Lu (BRAF V600E) and C8161 (BRAF wild-type) cell lines. Both were planted at two million cells into SCID mice, and the mice were treated with either vehicle control or 100 mg/kg PLX4720 by oral gavage twice daily. The results (Figure 4B and 4C) indicate that in the BRAF V600E tumors, PLX4720 caused significant tumor regression as there appeared to be little to no relative growth of the tumor, whereas in the BRAF wild-type tumor, PLX4720 appeared to have no effect. Furthermore, 1205Lu xenograft tumors were extracted from the mice, fixed in formaldehyde, paraffin embedded, and subject to immunostaining. Under immunostaining for P-ERK, the PLX4720 treated tumor extract indicated much lower P-ERK activity than the vehicle control. This indicates that PLX4720 not only selectively inhibits targets the BRAF V600E gene product in vitro (leading to tumor regression), but does so in vivo as well.

Fig. 4. Effect of PLX4720 on xenograft tumor growth. (A) Tumor volume measurements of COLO205 xenograft tumors treated with 5 or 20 mg/kg PLX4720 by oral gavage or treated with vehicle. Dosing occurred from days 1 to 14. (B and C) Two million cells [1205Lu (B); C8161 (C)] were s.c. injected into SCID mice. After reaching sufficient size, mice were treated by oral gavage with vehicle control (Left) or 100 mg/kg PLX4720 (Right) twice daily for the indicated times. (D) 1205Lu xenograft tumors were extracted, fixed in formalin, and paraffin embedded. Vehicle- (Left) and PLX4720- (Right) treated samples were immunostained for phospho-ERK.

Conclusion

In brief, the authors make the argument that PLX4720 (a compound they had discovered) selectively inhibits the BRAF V600E gene product. Upon inhibition, cell viability is decreased and tumor regression appears to occur in both cell and mouse models. This may have large clinical impact for cancer treatment as BRAF V600E is a common mutation known to occur in many cancers. This study may also shed light on the mechanisms of cancer inhibition as the BRAF-MEK-ERK pathway is shown to be critically down-regulated in BRAF V600E lines under PLX4720 treatment.

Critique

The authors of this paper went to great lengths to drive their point home through a variety of methods pointing to the same conclusion. This is not uncalled for, as the authors make a rather convincing argument using the substantial amounts of evidence obtained from several different experimental techniques. Readers are left convinced that PLX4720 appears to be a potent BRAF V600E inhibitor that will have a large impact on clinical cancer treatment. Indeed, this paper has been widely cited, and PLX4720 has been referred 4 times in the New York Times to date.

One thing to be desired from this study is justification of the concentrations and time-points at which PLX4720 was administered. The authors appeared to choose arbitrary values best suited to demonstrate their point. Not much thought had been given to the clinical relevance of such values. Since the end goal of PLX4720 is to be developed as an avenue of clinical cancer treatment, choosing clinically relevant concentrations is critical. For example, in the mouse model studies, the concentration of PLX4720 varied from as low as 5 mg/kg to as high as 100 mg/kg. There can be a significant difference in toxicity between such values. In future studies (perhaps closer to clinical phase II stage), it would be appropriate to address approximate toxicity levels for the inhibitor.

The use of the BRAF-MEK-ERK pathway to indicate inhibitor selectivity was never fully justified. It is only empirically observed that BRAF V600E mutants tend to be more dependent on the BRAF-MEK-ERK pathway. The authors provide some speculation such as failure of the negative feedback mechanism, but no evidence is ever provided. Moreover, the use of COLO205 over other V600E lines in mouse xenografts was also not justified. However, to be fair, justification of the BRAF-MEK-ERK pathway dependence is truly difficult and probably beyond the scope of this paper. Also, in the case of mouse xenografts, the authors supplement their study by using both BRAF V600E mutant and BRAF wide-type xenografts.

Cartilage engineered in predetermined shapes employing cell transplantation on synthetic biodegradable polymers

Woo Seob Kim, Joseph P. Vacanti, Linda Cima, David Mooney, Joseph Upton, Wolfgang C. Puelacher, Charles A. Vacan. Plastic and Reconstructive Surgery, Vol. 94, No. 2, pp. 233-237. August 1994.

Introduction During plastic and reconstructive surgery, inorganic materials are often implanted to provide support to produce the desired aesthetic or reconstructive effect. However, these inorganic materials are often recognized as foreign bodies by the body's immune system, leading to infection and ultimate rejection. If cartilage is to be safely and successfully implanted in patients, it must be immune-capatible. In this study, a team of researchers and medical doctors designed biodegradable polymers in a variety of shapes (e.g. triangle, rectangle, cross, cylinder), seeded them with chondrocytes, and observed the specimens during a 12-week time course. The chondrocytes were observed to take on the shape of their scaffolds with little evidence of resorption by the body or overgrowth beyond the bounds of the scaffold. These results suggest that autogenous cartilage, developed from a patient's own cells and grown on a pre-designed scaffold holds promise for use in the fields of plastic and reconstructive surgery.

Materials and Methods Researchers began with an entanglement of polyglycolic acid fabric (PGA), whose fibers are randomly arranged. This was then submersed in a 2% (w/v) solution of poly-L-lactic acid (PLLA), which chemically bonds the fibers of PGA by forming cross-linkages. After removal from solution, the polymer constructs were cut into the desired geometric shapes: a triangle,a rectangle, and a cross.

Cartilage was harvested from a bovine source and washed in povidone-iodine 10% solution. Chondrocytes were subsequently harvested and concentrated to 5x107 cells per mL. 100uL of this solution was seeded into the scaffolds and supplemented with 10% fetal calf serum and an assortment of amino acids and antibiotics. Cell nutrients were labeled with BrdU to allow researchers to identify implanted cells. The cell-polymer constructs were incubated in 5% carbon dioxide at 37 degrees Celsius, with the culture medium replaced every 3 days. After a week, the cell-polymer constructs were subcutaneously implanted into the dorsum of mice. For the cylindrical cartilage scaffolds, sheets of the polymer construct were cut into rectangles and seeded with chondrocytes that were incubated for a week. These polymer-cell constructs were then wrapped around 2.5 cm long silicone rods of 3mm diameter before being subcutaneously implanted into the dorsum of mice. After 3, 6, 9, and 12 weeks, mice were sacrificed and specimens evaluated with histological stains. Results and Discussion Gross examination of the non-cylindrical scaffolds (12 triangles, 12 rectangles, 12 crosses) showed that all scaffolds were replaced with chondrocytes that retained approximately the same shape as the scaffold. Two of the 12 cylindrical constructs became infected and were extruded through the skin -- the other 10 remained subcutaneous and showed replacement be chondrocytes, with retention of scaffold shape.

Histological staining showed that cells found on the periphery of the implants were more disordered and less mature than those located centrally, which exhibited deeper basophilic staining that suggests increased mucopolysaccharide concentration characteristic of mature cartilage. Immunohistochemical staining found BrdU labeling in the periphery of the cartilage, suggesting that cells located in this region were produced after in-vitro culturing. By week 3, the polymer fibers of the polymer-cell implants had begun degrading, with degradation continuing throughout the 12-week time course of the study.

Critique While this study represents a promising advance in creation and manipulation of autologous cartilage constructs, there are still several problems that must be addressed before this technique becomes feasible for use in human patients. To begin with, the time course of this study is relative short (12 weeks), whereas most cartilaginous implants are expected to last the duration of a patient's life. It is unclear if the shape of the cartilage may change past the 12 week mark; if the cartilage was to begin to lose its shape or lose its structural integrity, these implants would no longer be appropriate for use in plastic and reconstructive surgery. Researchers must also address the mechanical properties of cartilage formed on these scaffolds -- do they have the structural integrity of naturally-occuring cartilage? Can the mechanical properties be varied as a function of experimental parameters (e.g. density of injected cells, growth medium conditions)? This is an important aspect to address because cartilage used in different parts of the body may require different mechanical properties. A third problem the researchers may consider addressing is the immunoreactivity of their implants. It is interesting to note that in this study, chondrocytes harvested from calves were injected into scaffolds that were then implanted into mice. No mention of immune reactions is noted besides the two cylindrical scaffolds that were extruded. It is unclear if this immune reaction is due to the calf chondrocytes or perhaps the scaffold. Though 12 weeks is conceivably sufficiently long for an immune response to be elicited and noticed, long-term effects are unlikely to be noticed during the time course of this study. In conclusion, this paper presents a novel, perhaps revolutionary way for cartilaginous implants to be used in plastic and reconstructive surgery. However, before human implantation becomes even remotely feasible, researchers and medical doctors must take into account a host of factors that may influence the feasibility of such a design.

Tissue engineered autologous bladders for patients needing cystoplasty

http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T1B-4JN2NY2-2&_user=4420&_coverDate=04/21/2006&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1575238480&_rerunOrigin=scholar.google&_acct=C000059607&_version=1&_urlVersion=0&_userid=4420&md5=6d860a6cd88e8d22f5943ba9bc04b6c8&searchtype=a

OR

http://www.thelancet.com/journals/lancet/article/PIIS0140-6736(06)68438-9/fulltext

Anthony Atala, Stuart B. Bauer, Shay Soker, James J. Yoo, and Alan B. Retik

The Lancet, Volume 367, Issue 9518, Pages 1241 - 1246, 15 April 2006

Introduction and Background

Cystoplasty is a medical procedure used to surgically repair aspects of the bladder. It is commonly needed when medication is not enough to maintain organ function. Patients with myelomeningocele often require cystoplasty, as they experience frequent urination as often as every half hour. Traditionally, repair has been achieved by using two sources. Synthetic sources such as polyvinyl sponge, Teflon, collagen matrices, and, and biological sources such as the skin, omentum, placenta, and small intestines have been tested for use in cystoplasty. However, tissue grafts have often failed due to mechanical, structural, functional, and biocompatibility issues. Atala and his group have been working on a new system of reconstructing bladder tissue using the patient’s own cells. Their technique involves seeding scaffold with urothelial (bladder) and smooth muscle cells, incubating the construct in vitro, and implanting the organ into the patient.

Materials and Methods

The study and surgical technique was performed on 7 patients (range 4-19 years old) with severe bladder complications due to myelomeningocele. For each patient, urothelial and muscle cell samples were taken and cultured in vitro using standard techniques. Two types of scaffolds were made, one comprised of a decellularized bladder submucosa and the other consisting of a collagen-PGA composite. The bladder scaffolds were custom-made to match the sizes of the patient’s original organs, all with thickness at around 2 mm. The scaffolds were seeded with about 700 x 10⁶ cells of each type. The exterior was seeded first with muscle cells, incubated for 1 hour, and submerged in media that was changed every 12 hours. Next, the interior was seeded with urothelial cells, and the entire construct was incubated in media for 3-4 days when the surgery began.

During the procedure, the patients’ bladders were cut open, and the scaffold was oriented and attached in its place with fibrin glue and polyglycolic sutures. Some patients also had an omental wrap surrounding the construct. Urinary catheters were inserted during the surgery to allow for urine drainage during post-operative recovery. After a three week period, the bladder was conditioned by a cyclic process of clamping and draining through the catheters. Post-operative tests were done that included histological staining of the bladder, serum analyses, ultrasounds, leak point pressures, cystograms, and other urodymanic tests.

Results and Discussion

All the patients tolerated the procedure with no significant complications. Overall, bladder compliance and maximum leak-point pressures (indicating storage capacity) increased, showing a significant improvement with the engineered bladders. Histological tests confirmed proper growth of urothelial and muscle cells. The group found that the optimal technique was the use of a collagen-PGA construct that was inserted with an omental covering.

Critique

This study shows great promise in tissue engineering. The group has been testing tissue regeneration from autologous sources for quite a while, and the success of the group’s technique on human patients yields major implications for the future of tissue engineering. The relatively simple structure of the bladder even allowed them to tackle the challenge at an organ-level, but dealing with other organs will surely be more challenging.

The paper introduced many variables that were monitored, like the use of decellularized bladder submucosa versus a collagen-PGA scaffold, the effect of an omental wrap on bladder function, and the time intervals for cyclic conditioning of the engineered bladder. Performed with only 7 patients, there should be more replicates to fully determine which combination of techniques is optimal for bladder reconstruction. Also, although it showed an improvement in compliance and leak-point pressures, it fails to reference the ideal values that are typical for fully functional bladders. Moreover, the paper did not strongly address long-term factors related to the procedure. For example, it failed to mention the degradation behavior of the collagen-PGA scaffold, nor does it analyze the appropriate vascularization of the system. Thus, I believe more tests can be done to fully analyze the technique of autologous bladder reconstruction.

Tissue-Engineered Lungs for in Vivo Implantation

Thomas H. Petersen, Elizabeth A. Calle, Liping Zhao, Eun Jung Lee, Liqiong Gui, MichaSam B. Raredon, Kseniya Gavrilov, Tai Yi, Zhen W. Zhuang, Christopher Breuer, Erica Herzog, Laura E. Niklason1,

Introduction:

Human lungs usually do not repair beyond the cellular level, and currently the only method of replacing damaged lung tissue is through lung tissue transplantation, an expensive procedure that achieves less than 20% survival rate after ten years. Techniques to decellularize organs and use them as an acellular matrix scaffold to generate lung tissue have recently been tried with promising results. However, regenerating viable lung tissue is a tall order as the tissue should: contain lung specific cells, have branching structure of airways, separate blood and air, and allow for respiration. In an attempt to construct functional lung tissue using a rat model, lung tissue was decellularized and the resulting scaffold was repopulated with neonatal lung epithelial cells. The populated lung scaffold was cultured in a bioreactor simulating fetal lung environment. Lastly, the lung was tested for functionality.

Materials and Methods:

Preparation of a decellularized lung scaffold

Lung tissue from adult Fischer 344 rats was harvested and treated with detergent solution for roughly 2-3 hours. The resulting lung matrices, as verified by micro-CT scans indicate an intact microcellular matrix. The protocol was effective in preserving the micro structural properties of the native lung, yet removal of any antigenic components that may interfere with culturing of tissues.

Properties of the lung bioreactor

A bioreactor was used to culture the cells on the acellular scaffold. Cells were injected into the vasculature and airway compartments, to which they adhered. Subsequent tests indicated healthy proliferation and low incidences of apoptosis. Culture medium was fed though the pulmonary artery at physiological pressures and a negative pressure breathing loop with air was implemented to simulate respiration. The breathing loop was essential for ensuring cells did not block the airways and cleared the airways of any obstructions. In addition, exposure to air increased the numbers of epithelial cells, which was one of the essential criteria for a functional lung tissue graft. Compliance tests yielded high similarities between native tissue and engineered tissue, and an immunohistochemical staining indicated the seeded endothelial cells were extensively distributed with tight junctions between them. It was noted that extended culture periods resulted in a cellular distribution more similar to that of native lung tissues. In order to check if such scaffolding techniques are also compatible with human tissue, human lung tissue underwent a similar protocol of decellularization to obtain a matrix that was repopulated with lung cells. The cells adhered to the vasculature suggesting that such techniques may be compatible with human lung tissue, but an extensive study was not performed.

Implantation of engineered lungs into rats

The cultured engineered lungs were implanted and replaced the left lungs of four animals. Within minutes blood flow was established in the engineered lung vasculature. X-rays showed that inflation in the engineered lung was less than that of the right native lung. Additionally minimal bleeding was observed.

Results:

Data collected primarily consisted of testing of the engineered lung properties before testing for ability for gas exchange. Compliance testing for the elasticity of the engineered lung yielded similar stress-strain values between the native lung and the engineered lung. However, it was noted that the engineered lung was noticeably less elastic than the native lung; however, the difference was considered minor and did not alter the performance of the engineered lung.

Implantation of the engineered lung – Blood gas analysis of the blood taken from the engineered lung indicated that oxygen and carbon dioxide gas exchange did occur. Partial pressure of oxygen increased from 27 +/- 7mmHg in the pulmonary artery to 283 +/- 48 mmHg in the left pulmonary vein indicating hemoglobin oxygen saturation. However, this partial pressure is quite different from the native lung’s (634 +/- 69 mmHg. Additionally, levels of carbon dioxide exchange fell from 41 +/- 13mmHg in the pulmonary artery to 11 +/- 5 mmHg in pulmonary vein indicating efficient carbon dioxide removal.

Discussion

Overall, performance of the engineered lung was quite promising, although the tissue could not perfectly emulate that of native tissue. The engineered tissue overall was not as pliable and did leak some blood. However, performance was sufficient for the oxygenation of blood. Despite this, the success of the engineered tissue demonstrates a strong feasibility in the use of micro architecture of native lung tissue for regeneration of functional lung tissue. Still, many issues must be overcome for long term engineered function to persist. Ideally, the air-blood barrier must be improved to prevent further bleeding and production of certain proteins and surfactant needs to be increased. Lastly, other technologies for obtaining compatible epithelium such as lung stem cells is still required to make this a feasible clinical procedure.

Critique

The focus of this paper was to test the efficacy of the use of their engineered lung tissue as a functional tissue transplant. Considering that such functional lung tissue needs to emulate so many properties of native lung tissue in order to even function, and then for these researchers to be able to create such a working model is quite impressive. Based on the performance of the tissue, the researchers laid out clear goals to tackle in the future for improving the efficiency of the tissue. Aside from the mentioned technical improvements for tissue function, potential future projects include culturing of human lung tissues and improving incubation techniques.

However, there are several minor points in the paper that could be improved. Based on the protocol, a total of four engineered lungs were implanted into four rats. Because the researchers were simply testing the efficacy of the transplant, this number is adequate, although small. Future, improved engineered lungs could be tested with more rat models. Testing with human lung tissue was briefly mentioned but whether the cells could actually be cultured was not pursued. Given that this was not the purpose of the experiment, more tests need to be done before assessing whether human lung tissue could truly be created via similar techniques. Additionally, the researchers mentioned that the engineered lungs were functional for short periods of times, up to two hours, but little additional information was given on why this time frame was chosen. Whether the rats died or were simply euthanized after a period of time or that the tissues quickly lost efficiency is unknown. Lastly, much more qualitative data may be provided in terms of lung tissue properties. A small plot for the stress-strain characteristics of the tissues is provided albeit lacking details. However, the mention of reduced surfactant production and protein expression such as aquaporin-5 could be paired with relevant data.