Extracellular Pressure Stimulates Tumor Cell Adhesion In Vitro by Paxillin Activation

Citation:
J. van der Voort van Zyp, W.C. Conway, D.H. Craig, N. van der Voort van Zyp, V. Thamilselvan and M.D. Basson. Extracellular Pressure Stimulates Tumor Cell Adhesion In Vitro by Paxillin Activation. (2006). Cancer Biology & Therapy, 5 (9), 1169-1178.


Summary:
Tumor metastasis remains the most fatal complication of cancer, yet the mechanisms by which tumor cells are able to extravisate and re-attach to the endothelial lining of remote blood vessels is poorly understood. This paper explores the role that paxillin, which is involved in cytoskeletal regulation and growth, plays in mediating tumor cell adhesion to extracellular matrix. Adhesion of cells had been previously shown to be enhanced by pressure-induced (15 mmHg) phosphorylation of the focal adhesion proteins FAK and Src. In addition, studies have shown that the activity of FAK and Src is linked to the cytoskeleton. However, the mechanism by which these are linked has been poorly characterized. Paxillin is a protein which is known to interact with both cytoskeletal proteins and focal adhesion proteins such as FAK and Src. The authors thus theorized that paxillin might have been the link between these events.

The authors first were able to show that increasing the extracellular pressure increased the phosphorylation of paxillin, and noted an increase in the binding ability of cells versus the control sample. Then, by using small interfering RNA (siRNA) to suppress the expression of paxillin, the authors were able to negate the pressure-induced increase in adhesion observed previously. The authors then investigated the effect of inhibited paxillin expression on the activation of FAK by phosphorylation at the 397 tyrosine residue. Inhibition of paxillin expression was shown to have no effect on FAK 397 phosphorylation under normal conditions, but did inhibit the pressure-induced phosphorylation observed in control samples. Similar results were observed for Src 416 phosphorylation. Blocking of FAK and Src activity by other means was not shown to have any effect on paxillin expression or phosphorylation.

The authors conclude that paxillin is an important intermediary in inside-out adhesion signalling, being somewhere upstream of FAK and Src. Other studies have shown that in addition, paxillin may be phosphorylated by FAK in outside-in adhesion signalling. The authors speculate that this difference in signaling direction may be caused by the actual adherence event; suspended cells exhibit inside-out signaling which mediates adhesion, while adherent cells exhibit outside-in signaling.

Significance:
Most fatal cases of cancer are the result of tumor metastasis. Understanding the mechanisms by which tumor cells are able to enter the bloodstream and adhere to remote locations to establish a secondary tumor can help in the development of effective treatments to prevent the metastasis of tumor cells. Although the mechanism of paxillin inhibition presented in this paper is ineffective in a clinical setting, this paper presents us with another possible route to diagnose and treat uncontrolled tumor cell metastasis.

Yeast Life Span Extension

Yeast Life Span Extension by Depletion of 60S Ribosomal Subunits Is Mediated by Gcn4

Kristan K. Steffen,Vivian L. MacKay,Emily O. Kerr,Mitsuhiro Tsuchiya,Di Hu,Lindsay A. Fox,Nick Dang,Elijah D. Johnston,Jonathan A. Oakes,Bie N. Tchao,Diana N. Pak,Stanley Fields,Brian K. Kennedy,andMatt Kaeberlein.

Cell, Volume 133, Issue 2, 292-302, 18 April 2008

 

Summary:

It is well known that reduced caloric intake leads to the extension of life span.  For yeast, dietary restriction is mediated by three kinases, that are particularly responsible for mediating various cellular processes such as: stress response, protein turnover, cell growth, and ribosome biogenesis.  This research focused on how the reduction of the level of 60S ribosomal subunits in the cell will slow aging in yeast.  The researchers studied this by using various methods of inhibiting the activity of the 60S subunits with a small molecule inhibitor, or by deleting the gene that codes for the subunit or its processing factors.  These three methods were all sufficient in increasing the life span of the yeast cells.

The life spans were measured by graphing the fraction of viable cells after every generation.  The normal yeast cells typically had relatively good viability until the ~25th generation, whereas when under dietary restriction, a large majority of the cells were still viable until the ~35th generation.

This research showed that the cells that with a long life span due to the decrease in 60S subunit levels, had an increased amount of Gcn4 activation.  Gcn4 is a nutrient-responsive transcription factor.  This factor we required for the cells to have the increased longevity.

It is particularly interesting to note that decreasing the amount of 40S subunits has no major effect on cell longevity.  Also, the basic molecular mechanism discussed in this paper has also been observed to exist in small animals and insects.  Therefore, the authors suggest dietary restriction, protein translation, and longevity are linked and are evolutionary conserved.

 

Significance:

This paper discusses a particular molecular mechanism that contributes to why dietary restriction will increases cell longevity.  The link between caloric intake and life span extension is not fully understood yet, and this was an attempt at identify a possible molecular process that is involved in this process.  However, additional information about the cause of cell death was not thoroughly discussed.  For example, one theory is that cells die because of the accumulation of cellular junk, which when above a particular threshold level, becomes pathological.  With that theory in mind, it would be easy to predict that reducing the level of 60S ribosomal subunit will almost certainly extend life, because less 60S ribosomal subunit means that less translation can occur, which means that there is less stuff that could potentially become cellular junk (i.e. undigestible proteins).  This is also an important factor in regards to the 3 kinases that mediate the dietary restriction, because they are involved with various metabolic cellular processes.  In other words, it is expected that anything that would slow down the rate of metabolism, would also slow down the rate of intracellular junk buildup, which means that it would take longer for the junk buildup to cause pathology.

Further studies into why the 40S subunit have no major effect on cell longevity would be extremely interesting, because this is the only factor that may contradict the theory of cell death.  Also, it would be interesting to know the effects of increasing the intercellular junk collection rate on longevity in cells with and without the decreased 60S subunit level.

Reprogramming of murine and human somatic cells using a single polycistronic vector

Bryce W. Carey, Styliani Markoulaki, Jacob Hanna, Kris Saha, Qing Gao, Maisam Mitalipova

PNAS, January 6, 2009, Vol. 106, no. 1, 157-162


Summary

Typically, generation of induced pluripotent stem cells (iPS) requires multiple proviral integrations to acheive viral-based delivery of the reprogramming factor; multiple proviral integrations pose the danger of insertional mutagenesis. This paper suggests a new approach to reprogram human somatic cells into patient-specific stem cells using just a single vector. Authors used 2A "self-cleaving" peptides which support efficeint polycistronic expression from a single promotor to deliver reprogramming factors in a single virus. They found four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) can be expressed from a single virus to generate iPS cells in both embryonic and adult somatic mouse cells. Additionally, human induced pluripotent stem cell (hiPS) lines from human keratinocyts were generated using the same approach.


Key Concept

• 2A "self-cleaving" peptides - 2A peptides originally identified and characterized in apthovirus foot-and-mouth disase virus. 2A oligopeptides contain a higly conserved c-terminal D(V/I)EXNPGP motif that mediates "ribosomal skipping" at the terminal 2A proline and subsequent amino acid. They used F2A, T2A and E2A peptides derived from different viruses.


• Single polycistronic vector - This vector can translate several reprogramming vectors from one promoter using 2A peptides. In this study they made 4F2A vector which contains Oct4, Sox2, Klf4 and c-Myc. Three different 2A peptides seperate those four factors when they are translated.
• Doxycycline (DOX)-inducible lentivirus - A tetracycline inducible lentivirus vector was constructed where expression of the genes was controlled from the tetracycline operator minimal promoter (tetOP) They, first, added DOX to generate four reprogramming factors. Once the somatic cells were transformed into iPS cells, they stopped adding DOX to check that iPS cells show 'stemness'; if iPS cells contain 'stemness', cells will renew themselves automatically even without external expression of reprogramming factors.


• Nanog GFP - The expression of Nanog is used as a key limestone to ensure the stemness of iPS cells. Green flourescent protein was attaced to nanog and used as a marker.


• Three germ layer - Endoderm, Ectoderm, Mesoderm were used as key markers for stemness of iPS cell line.

Significance

• Multiple technologies may converge to allow the generation of therapeutically acceptable iPS cells without viral integrations or lingering reprogramming factors
• Because the four factors are expressed from a defined location, the polycistronic vector system should simplify the study of reprogramming mechanism.

Prevention of Cartilage Degeneration in a Rat Model of Osteoarthritis by Intraarticular Treatment With Recombinant Lubricin

Carl R. Flannery, Richard Zollner, Chris Corcoran,Aled R. Jones, Adam Root,Moise´s A. Rivera-Bermu´dez, Tracey Blanchet, Jason P. Gleghorn, Lawrence J. Bonassar, Alison M. Bendele, Elisabeth A. Morris, and Sonya S. Glasson

ARTHRITIS & RHEUMATISM
Vol. 60, No. 3, March 2009, pp 840–847
DOI 10.1002/art.24304

Summary

Osteoarthritis is a degenerative disease characterized by the progressive loss of articular cartilage that affects the quality of life of millions of people around the world. Lubricants provide a crucial role in the protection of the articular cartilage by providing a friction-resistant coating to cartilage surfaces in joints. Lubricin is one of the glycoprotein in the lubricant that is proven to provide a significant role in preventing cartilage wear, synovial cell adhesion, synovial cell proliferation. Because mutations in the Lubricin gene are known to cause osteoarthritis (OA) symptoms, the paper reasoned that increase lubricin levels will be a good therapy for OA. Thus, the overall goal of the paper is to study the efficacy of injecting a novel recombinant of lubricin into rats with OA.

The generation of a novel recombinant of lubricin, instead of using the full gene sequence was done to optimize the production yields. The recombinant was created by exposing cDNA for human lubricin with restriction enzymes to replace the repetitive elements with a synthetic cassette (Figure 1a). The resulting construct was called LUB:1 and was transfected and purified from hamster ovary cells. LUB:1 protein was purified through affinity and size-exclusion chromatography, analyzed by SDS-PAGE gels, and are either stained with Coomassie blue (Lane 1 in Figure 1B) or transferred onto nitrocellulose membranes for Western blotting (Lane 2 in figure 1b)

Figure 1: A) a graphic representation of the relationship between Lubricin gene and the construct. B) Coomassie blue stains and Western Blotting after non-reducing SDS-PAGE. (Triangles indication the monomer and dimer of LUB:1. C) shows resistance of LUB:1 to digestion of neuraminidase (Land 3) and endo-O-glycosidase, but not both combined.


To test LUB:1 binding, bovine cartilages were extracted, native lubricin removed, specimens incubated with either LUB:1 or just PBS, and immunostained (Figure 2a). To test whether LUB:1 reduces friction, Bovine cartlage were exposed to either PBS alone or with increasing concentrations of LUB:1 and tested on a custom testing apparatus.

Figure 2: A) Bovine cartilage has an absence of lubricant binding. B) Arrows show the presence LUB:1 binding. C) Friction of coefficient is significantly lower with presence of LUB:1. D) Increase LUB:1 lowers cell adhesion



20 rat models had cartilage tears induced through surgery. One group of rats received doses of PBS. The other groups received intraarticular doses of LUB:1 either once a week or 3 times a week. Doses of albumin injections were used as a control for a group. After 4 weeks, rats were killed and OA was scored on cartilage degeneration, total joint score, width of severe lesions, significant cartilage degeneration width, bone score, and medial capsule width.

Figure 3: The analysis of histologic sections of the knee joints reveal score reductions throughout all categories with the presence of LUB:1, especially severe lesions width where there is a 83% decrease.





Figure 4: Rat joints with OA were treated with either PBS or LUB:1 3x a week. The figure shows that LUB:1 were able to preserve the cartilage structure, whereas the PBS-treated (4A) has an arrow indicating severe cartilage lesion.


Significance:
Since cartilage degeneration plays a major role in the progression of OA, studying therapies that preserve the structural integrity of the cartilage will lead to a reduction to a major cause of OA, resulting in an increase in mobility and quality of life in patients. Current treatments include anti-inflammatory drugs and inhibitors that provide symptomatic relief but do not prevent cartilage degeneration. Treatment strategies to inhibit collagenase have low efficacy and/or harmful side effects. This paper shows an novel approach to OA therapy using a higher yielding LUB:1 construct that produces significant results across most OA scores in rat models.

A hypothesis for cell aging in mature adipocytes

Chronological changes in metabolism and functions of cultured adipocytes: a hypothesis for cell aging in mature adipocytes

Yi-Hao Yu and Huaijie Zhu
Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032
Submitted 3 June 2003; accepted in final form 13 November 2003

SUMMARY
Yu and Zhu investigated chronological changes in metabolism and function of adipocytes in vitro. In a tissue culture system, they grew 3T3-L1 preadipocytes, which were then induced to differentiate as adipocytes. They characterized several factors as variables of cell age. They observed that as adipocytes aged, there was a corresponding elevation in the expression of aging markers Bax and lipopolysaccharide-induced TNF-_ Factor (LITAF). These two proteins play a role in the proapoptotic scene. The researchers also observed a decrease in fuel uptake and overall cellular catabolism as cells aged. Glucose uptake decreased, as did the CO2 production rate (measure of cellular respiration rate) during adipocyte aging (CO2 production initially increases and stabilizes after differentiation; the decrease is observed in relation to this stabilized value at maturation). Catabolic activities were also characterized; as cells aged there was a decrease in insulin sensitivity, as well reduced gene expression of secretory proteins adiponectin and leptin. While these observations of cell aging were a cell culture phenomenon, this data suggest that similar characteristics of adipocyte aging, namely being more metabolically inert, may be found in vivo.

WHY I CHOSE THIS PAPER
By characterizing the observed metabolic changes that occur during adipocyte aging, this paper provides a baseline with which to measure cell aging in a specific cell line in vitro. I am interested the SENS platform for reversing cell aging, particularly the role of "extracellular junk." This baseline allows us a method to measure cell age in our experiments. While characteristics of an observed cell aging process cannot necessarily and simply be turned into indicators of cell vaibility and youthfulness, it is perhaps a start in my search for a way to gauge the slow or reversal of cell aging in an in vitroexperiment for this class

Inhibitory effects of nitric oxide on invasion of human cancer cells

Citation:
Wang, F., Zhang, R., Xia, T., Hsu, E., Cai, Y., Gu, Z., Hankinson, O. Inhibitory effects of nitric oxide on invasion of human cancer cells (2007). Cancer Letters, 257 (2), pp. 274-282.
PMID: 17869411

Summary:
This paper talks about testing whether or not NO had an inhibitory effect on cancer cell invasion and also exploring the mechanism of the inhibition. In particular, they explored the effect of NO on HIF-1, which is involved in the transcription of many genes linked to cell proliferation, angiogenesis, and metastasis. HIF-1 often overexpressed in human cancers as a result of hypoxia at the tumor site and genetic mutations; and its overexpression has been associated with patient morality in many studies.

For their experiments, the group used prostate cancer cell line PC-3M and bladder cancer cell line T24 cells as their strains and performed invasion assays using Matrigel invasion chambers. The cells were seeded in the Matrigel chambers both under normal conditions and also after hypoxia treatments , during which the chambers were placed in an airtight cell culture incubator maintained with low levels of oxygen for 24 hours and then cultured under normal conditions afterwards for another 24 hours.

In their experiments, sodium nitroprusside (SNP) was used as a nitric oxide donor. In addition to invasion assays, immunoblotting techniques were used to detect HIF-1 protein levels and cell proliferation rates were measured for cultures treated with SNP alone and with SNP and a NO scavenger PTIO together, as well as other controls. From the experiments, hypoxia conditions increased cell proliferation and invasion as well as higher levels of HIF-1. Their results also showed that SNP strongly inhibited the invasion of the cells. In the presence of a NO scavenger), the effect of SNP on invasion was negated, which suggests that invasion is dependent on how much NO is released. From the immunoblotting assays, it was shown that SNP inhibits the expression of HIF-1 for the two strains at 100uM.

The group has concluded that the inhibitory effect of NO is related to inhibiting HIF-1. However, they do no believe that this is the only mechanism since some of their results could not be explained fully. They also hypothesize that NO inhibits the function of mitochondria and that mitochondria inhibitors reduces the invasion of cancer cells.

Significance:
In clinical applications, hypoxia is associated with cancer cell resistance to chemotherapy, metastasis and patient mortality. Since most cancer related deaths are a result of uncontrollable metastasis of tumor cells, mechanisms by which cancer cells invasion can be inhibited would be useful in predicting and designing drugs that target the mobility of cancer cells. The discovery of the inhibitory effects of nitric oxide on cancer cell invasion suggests that NO donors may be effective cancer chemotherapeutic agents. Since its mechanism for inhibiting cancer cell motility is related to inhibiting HIF-1, other ways of down-regulating the effect of HIF-1 in cancer cells or reducing hypoxia at cancer sites can also be explored for their therapeutic potential.

Fibroblast sheets co-cultured with endothelial progenitor cells improvecardiac function of infarcted hearts

Authors:
Hiroshi Kobayashi, MD · Tatsuya Shimizu, MD, PhD
Masayuki Yamato, PhD · Kayoko Tono, MS
Haruchika Masuda, MD, PhD
Takayuki Asahara, MD, PhD
Hiroshi Kasanuki,MD, PhD · Teruo Okano, PhD

J Artif Organs (2008) 11:141–147

The study was done in an effort to improve existing therapies for better cardiac function post heart attack or other related injuries (post attack, usually ventricular function declines). Namely, the focus has been on treatments involving regenerative medicine. Echoing other contributors of the blog, heart problems remains the leading cause of death and a main concern in the present day population. With new and better ways to treat injuries, we can prolong lives and improve life quality post a (serious) heart injury.

Brief Materials Used:
Animal model used: male athymic rats (8-10 weeks old); induced myocardial infarction by ligating the left anterior descending coronary artery so that blood flow ceases. Guinea pigs are another possible model that could have been used. Their hearts are similar to humans (with electrical conduction system and similar muscle contraction system) and bigger than rats or mice. (ref: http://scienceandresearch.homeoffice.gov.uk/animal-research/publications-and-reference/001-abstracts/abstracts2-2006/04september-2006/373?view=Html). The endothelial progenitor cell (EPC) cultures were made from peripheral blood mononuclear cells from Sprague-Dawley rats; fibroblast sheets were cultured from abdominal skin samples from Wistar rats. Refer to article for details of the cell density, time of culture, temperatures, etc.

Experiment:
All rats underwent the induced infarction. Then they were randomly put into four equal sized (n=5) groups: one control who received transplant operation but no transplant (C group), one group received only EPC (E group), one group received only triple-layer fibroblast sheets (F group), and last group received the sandwich-like construct of sheets+EPC (E+F group).

Echocardiographies before transplantation and then weekly for a month were used to measure rat cardiac function. And the animals were sacrificed at the end of the period for histological analyses. Statistical analyses proceeded to evaluate the data. Examples of variables investigated include left ventricular dimensions/wall thickness, quantity of connective tissue via staining. Left ventricular performance (measured by echocardiograph) had significant improvement one week after transplation (and in weeks 2, 3, 4) in the E+F group compared to C group. The other two groups also showed better results than control but not significantly different compared to E+F group. Vascularization in the E+F group was better than E and C group. The vessel walls included both EPC and fibroblasts for the E+F group, suggesting higher effectiveness in transplantation of EPC+Fibroblasts than either two alone. The combination of EPC and fibroblast sheet allows for better adhesion (single cells can and do wash out easily), and accelerate vascularization (with combination of EPC and growth factors embedded between the sheets). Previous studies with fibroblast sheet alone or endothelial cells alone have shown that combination therapy is, in fact, an improvement.

Although the data gathered for this particular paper doesn't show significant improvements from the combination therapy compared to the individualized therapies, the authors referred to results from many other similar and applicable previous experiments, justifying further investigation (using larger animal models, optimizing culturing period and transplant timing, larger sample size, perhaps older animals, etc.). The variables they measured and calculated correlated with cardiac functionalities to address the problem at hand.

A microfluidic culture platform for CNS axonal injury, regeneration and transport

Anne M Taylor, Mathew Blurton-Jones, Seog Woo Rhee, David H Cribbs, Carl W Cotman and Noo Li Jeon

Nature Methods 2, 599 - 605 (2005)

Summary:

This group has devised a microfluidic culture system that allows growth of central nervous system (CNS) neurons into compartments that separate axons from its somata and dendrites. Their microfluidic device allows observation and experimentation on the axon region of the neuron. This device was used to observe how the neuron responded to damage resulting from axotomy. Other experimental parameters were investigated to ensure validity of their methods.

The device contains two regions where one houses the somal part of the neuron while the other offers room for axonal growth. Dividing the regions were microgrooves which allowed the axon to grow into the axonal region while the cell bodies stayed in the somal side of the device. A slight volume difference between the two sides caused fluid to constantly flow into the axonal side in order to fluidically isolate the axonal regions of the neuron from the rest of the device. The microgrooves and volume difference served to keep the fluid in the axonal side from diffusing back into the somal side.

Experiments performed include verification that the axonal side was isolated from the somal side and observations on neuronal response to axonal damage. Texas red dextran, a fluorescent dye, was administered in the axonal side. After twenty hours, the somal side did not show traces of this dye, presenting effective isolation of the two regions. [35S] methionine (131 Da) was also successfully confined to the axonal side for over twenty hours to further demonstrate isolation of the axonal side from the rest of the device.

After isolation verification, neurons were grown where their axons were separated from the dendrite regions with barriers in the device. The placement of the barriers was assessed with immunocytochemistry as the neuron matured in order to keep the axons isolated from dendrites at different stages of neuron development. The group also isolated axonal mRNA, and using reverse transcriptase- polymerase chain reaction (RT-PCR), they found that in the mRNA of developing axons, coding for synaptophysin or the presynaptic vesicle protein was present.

Vacuum aspiration was performed on the axonal side for five seconds to damage the axons. Again, RT-PCR was used to observe mRNA activity occurring in the somal side of the device. Upon response to axonal injury, the somal side expressed FBJ murine osteosarcoma viral oncogene homolog or c-fos mRNA in large amounts, demonstrating immediate recovery activity. A final experiment was administration of BDNF and NT-3 for five days to an experimental batch of injured axons while a control batch didn’t receive any neurotrophins. The result was tremendous growth in the axons for the neurotrophin-treated batch compared to the control batch.

Significance:

The methods and devices used prior to this group’s work such as Campenot chambers failed to culture CNS neurons. Campenot chambers were excellent in culturing peripheral neural system neurons using either nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF). However, these devices weren’t able to culture CNS neurons because CNS neurons didn’t mainly depend on neurotrophins for growth and are generally more difficult to culture. The Campenot chambers were also leaky at times.

The microfluidic device that this group created overcame these problems and has cultured rat cortical and hippocampal CNS neurons. This group innovatively cultured CNS neurons, effectively kept axon regions isolated, and successfully experimented on the recovery activity of axons. They are also first to report found that coding for synaptophysin or the presynaptic vesicle protein was present in the mRNA of developing axons.

Antithrombogenic property of bone marrow mesenchymal stem cells in nanofibrous vascular grafts

Craig K. Hashi, Yiqian Zhu, Guo-Yuan Yang, William L. Young, Benjamin S. Hsiao, Karin Wang, Benjamin Chu, and Song Li

PNAS  July 17, 2007  vol. 104  no. 29  11915–11920

Summary:

This study shows an effective method for producing new antithrombogenic cardiovascular grafts. The material used is  is a synthetic polymer that is to be electrospun into a cylindrical shape. These nanofibers are advantageous for cardiovascular grafts because cells can be seated very well on top. 

Looking at the picture on the left, b is the electrospun nanofibers and a,c, and d are images of actual vessels. As you can see, the natural vessels are all aligned in one direction. This is another advantage of the nanofiber grafts, they are able to be aligned similarily to resemble real live vessels. In e, mesenchymal stem cells are seated on the nanofiber and then wrapped around to form a cylindrical structure. 

In vivo studies, the researcher implanted nanofiber grafts with and without MSCs seeded on the grafts. The picture on the right shows the histology samples of the implanted grafts. The ones on the left are the grafts without any MSCs 60 days after implantation and the pictures on the right are the grafts with MSCs. It is noticable that there is much less thrombin buildup in the graft with MSCs. 

Significance:

For the longest time, the holy grail of cardiovascular bioengineering is the ability to produce artificial vessels without the buildup of thrombin along the inside walls. This is a big problem because if blood vessels become damaged, there would be no effective way to transport blood in a manner that closely resembles that of a natural body. There are numerous vascular grafts on the market right now that have a patency of roughly 50% for the next few years that it is implanted into the body. This is obviously not acceptable when the probability of survival of the graft is nothing more than a coin flip. This study made a big impact in the cardiovascular field by implanting mesenchymal stem cells onto the inside walls of nanofiber grafts and showing that these cells do have the ability to prevent thrombin buildup and vessel clogging. And on top of that, these grafts are able to be manufactured at much smaller diameters than current grafts on the market, allowing for more flexibility of vessel sizes depending on the patient.  

HIV-1 Nef triggers Vav-mediated signaling pathway leading to functional and morphological differentiation of dendritic cells

MARIA GIOVANNA QUARANTA,* BENEDETTA MATTIOLI,* FRANCESCA SPADARO,* ELISABETTA STRAFACE,† LUCIANA GIORDANI,* CARLO RAMONI,* WALTER MALORNI,†AND MARINA VIORA*,1

*Department of Immunology, †Department of Ultrastructures, Istituto Superiore di Sanita`, 00161 Rome, Italy

Summary

Regulated migration of dendritic cells (DC), which is primed by different transcriptional factors leading to modulation of genes, plays a central role in induction of physiological immune responses and this process necessities plasticity of cytoskeleton. DCs are the first target of HIV and, by clustering and activating T-cells, may both activate antiviral immunity and facilitate virus dissemination. HIV-1 Nef protein is expressed early during infection and has been shown to be critical for viral pathogenesis in vivo, enhancing viral replication and infectivity. Depending on its intracellular localization, Nef interferes with cellular signal transduction pathway, also exogenous Nef inhibits the induction of a specific antibody response, participating in AIDS pathogenesis. In this paper, authors investigated Nef-induced DC differentiation, focusing on the interference of Nef in the signaling pathways that regulates DC maturation. They found that exogenous Nef enters immature DCs, targets Vav, activating its signaling cascade via Rac1, leading to cytoskeleton rearrangements, leading to cytoskeleton rearrangement and NF-kB activation.

Following information contains brief description of what was done during research experiment. Recombinant HIV-1 Nef protein was obtained from E.Coli. The Nef uptake by DC was elaluated by flow cytometry using FITC conjugate Nef. CLSM and IVM analysis were used to investigate the subcellular localization of Nef in the mature DC; kinetic analysis was carried out to analyze whether Nef signaling influences subcellular Vav distribution. NF-kB p65/NF-kB p50 transcription factor kits were used to analyze the rates of NF-kB activation from total lysates from untreated and Nef – or LPS-treated DCs. Nef induced morphological changes and cytoskeleton and vinculin rearrangements which were tested by SEM. Using DC/T cell conjugate analyses and fluorescent staining, it was testified that Nef improves immunological synapse formation between DCs and CD4+ T cells. Nef also enhanced nucleotide exchange rate of Rac1 and Cdc42 which was analyzed through particular protein fusion.

Summary of results is depicted in the figure:

Figure 8. Signaling events in Nef-induced DC maturation. Nef enters immature DCs leading to Vav phosphorylation. This is achieved by either direct interaction with Vav or an upstream activation of Src kinases, which in turn phosphorylate Vav. P-Vav is then able to activate Rac1 and Cdc42 proteins that trigger a series of downstream signaling events. Those include rearrangement in the actin and vinculin cytoskeleton, leading to morphological changes, and NF-kB translocation to the nucleus, which yields DC phenotypical and functional differentiation. In this scenario, Nef changes the local environment of the DCs resulting in their activation.

Significance:

Considering that mucosal DCs and blood DC-SIGN⁺ DCs represent the first HIV-1 targets upon sexual transmission and transmission via blood, in this study immature DCs are used as the closest model for in vivo primary HIV infection to investigate the effect of Nef on DC maturation. This work outlines Nef induced, Vav-dependent signaling pathway by which HIV-1 may take advantage of DCs favoring non-specific T cell activation, permitting immune system invasion that leads to AIDS. Understanding of the virus invasion mechanism is a necessary key to creation of effective treatment with knowledge of the steps that can be blocked in that pathway. The Nef-induced signaling pathway regulating DC functional and morphological differentiation could provide opportunities for therapeutic manipulation of immune system responses in vivo.

Article can be found on this link: http://www.fasebj.org/cgi/reprint/17/14/2025.pdf

Induction of heat shock protein 70 protects mesangial cells against oxidative injury

Hung-Chun Chen, Jinn-Yuh Guh, Juei-Hsiung Tsai, and Yung-Hsiung Lai. Kidney International, Vol 56 (1999) pp. 1270-1273

Summary:

When cells are put under elevated temperatures or other types of stress and injuries, Heat Shock mRNA and Protein expressions are upregulated. There are several types of heat shock proteins (hsps) and they are categorized by their mass in mammals. Hsp60, hsp70, hsp90 are studied most often in human. They protect cell function in several ways. They are chaperones for other proteins and ensure the correct conformation by stabilizing partially folded proteins. They also provide extra transport mechanics for tagged proteins to be destroyed. Scientists believe more functions are yet to be discovered. Recent studies show that oxidative stress can also induce hsp expressions. This is important because oxidative stress occurs during mesangial injury. Mesangial cells are specialized cells around blood vessels in kidneys, at the mesangium. This paper explores the effect of oxidative and heat shock stress in mesangial cells.

The methods used in this paper resembles the techniques in BE115. Glomeruli cells were harvested with trypsin and cultured in media similar to ours. However, cells were starved for 24 hours before the experiment is conducted. Heat shock is induced by incubating the cells in 45C for 15 minutes. Oxidative stress is induced by exposing the cells to xanthine and xanthine oxidase. As negative controls, some cells are just incubated with xanthine or xanthine oxidase alone. The results are measured via Thymidine uptake, trypan blue exclusion method, and western blot. Thymidine measures how fast the gene is transcribed and trypan blue measures what percentage of cells are alive. If cells are just stressed under heat or incubated with xanthine or xanthine oxidase alone, thymidine uptake and trypan blue exclusion yielded same result as the control group - high survival rate. In contrary, oxidative stress lowered cell survivability to 35%. However, cells which were previously exposed to heat shock then oxidative stress had higher rate of survival (84%). Third method to measure result was western blot, which can quantify how much hsp70 mRNA and protein are present. Result shows hsp is only produced when cells are heat shocked. In no other case do the cells exhibit upregulation in hsp70 mRNA and proteins.

Significance:

The result indicates that hsp is only upregulated when heat is applied to the cells and hsp do not respond to oxidative stress. Although oxidative stress does not induce hsp expression, cells are protected from oxidative stress if there are hsp present in the cell. Since mesangial cells undergo oxidative stress during injury, which further causes cell deaths, manipulating heat shock proteins may ease the cell mortality rate.

Dynamics of the Self-Assembly of Complex Cellular Aggregates on Micromolded Nonadhesive Hydrogels

Dynamics of the Self-Assembly of Complex Cellular Aggregates on Micromolded Nonadhesive Hydrogels

Summary:

Napolitano, A. P., Chai, P., Dean, D. M., Morgan, J. R. (2007) Dynamics of the Self-Assembly of Complex Cellular Aggregates on Micromolded Nonadhesive Hydrogels. Tissue Engineering, 13(8), 2087-2094. doi:10.1089/ten.2006.0190

Self-assembly of cells has been observed in both embryonic tissues and adult cells. Spheroids are formed that mimic in vivo differentiation and cellular interactions (cell-cell and cell-extracellular matrix (ECM)). Large tissue-engineering structures are partly dependent on self-assembly, so knowing the dynamics of assembly is important.

Napolitano, et al. developed a method to form cellular aggregates on micromolded nonadhesive hydrogels for this purpose. Polyacrylamide hydrogels and agarose gels were cast using a wax mold and incubated in medium. Normal human fibroblasts (NHFs) and human umbilical vein endothelial cells (HUVECs) were cultured in the recesses of the gels.

They found that cells sank into recesses within an hour and spheroids formed within 24 hours. Aggregate geometry and size was dependent on recess geometry as well as density of cells. The spheroids could also be easily removed by inverting and briefly centrifuging. The use of a hydrogel substrate minimized cell-substrate interactions so that the neighboring cells would determine the environment.

Using mixed cell suspensions of NHFs and HUVECs, the researchers were able to demonstrate that not only do the cells self-assemble into layered spheroids but also that mature spheroids can reorganize to incorporate additional cells that are added later to form the same layered spheroids.

The authors indicate that their experimental data supports cytokeleton-mediated (adhesion and interfactial) tension and cell contraction playing a role during self-assembly

Significance:

An understanding of the forces in play when cells aggregate is important for designing tissue-engineered devices. This type of spheroid production has potential for transplantation if it can be scaled up or applied to more complex geometries.

Generation of Tubular Superstructures by Piling of Renal Stem/Progenitor Cells

Will W. Minuth, Ph.D., Lucia Denk, Tec., and Hayo Castrop, Ph.D.
Tissue Engineering: Part C, Methods. Mar 2008; 14(1): 3-13.

Summary
With hundreds of thousands of Americans suffering from various stages of renal failure, there is a great deal of interest in potential mechanisms of kidney regeneration. Under appropriate perfusive conditions, rabbit renal stem and progenitor cells have been coaxed, in vitro, into basic tubule formations that exhibit protein markers and structural characteristics representative of natural renal tubules. Whether they sufficiently exhibit the functionalities of said tubules remains to be investigated, but researchers hope this development of techniques that result in relatively large scale, coordinated production of tubule superstructures makes tissue engineering a likely player in future treatments for kidney failure.

In this experiment, researchers stripped the fibrous lining from harvested newborn rabbit kidneys and sandwiched this embryonic renal tissue in between thin sheets of polyester fleece. These arrangements were then enclosed in tissue holders and exposed to constant horizontal flow of fresh media.

Image 1: Cross-sectional illustration and scaled view of the culture subunits.

A solution of IMDM with an antibiotic-antimycotic cocktail and aldosterone (for promotion of tubule growth) was perfused through the tissue culture continuously for 13 days at 1mL/hr and 37°C. The developed tubules were fluorescently labeled with Soybean Agglutinin for analysis under microscope as whole-mount specimens and as cryosections, as well as labeled with fluorescent antibodies for detection of various other protein markers.

Ultimately, the cultured tubular structures exhibited cells arranged directionally, a visible inner channel (lumen), and ECM proteins consistent with an epithelial basal lamina—each of which are characteristic of natural renal tubules. The tubules also showed reaction with numerous markers for proteins, such as Na/K-ATPase and Fibrillin-1, that were not found in untreated embryonic cells—suggesting significant differentiation. While the exact mechanisms for growth of these tubules from single cells remains unknown, it is clear that the combination of mechanical and molecular conditions in this perfusion setup produces some promising results.

The straightforward preparation of these arrays of tubules sets forth a number of further investigations into this technique. Concerned with scalability of production, the researchers of this study performed subsequent trials in which they piled (stacked like bricks) and paved (laid like tile) several of these cell culture ‘sandwiches’ in the same chamber. The amount and degree of cell differentiation in these chambers was nearly identical to that in the single-culture chambers, and these tubules displayed the same characteristic morphologies and protein markers.

Image 2: Top: Cross-sectional views of developed tubules, exhibiting a basal lamina (asterisk) and marked lumen (arrow). Middle and bottom: Illustrations of piled and paved arrays for generation of renal tubule superstructures and the resultant perfusion of growth media.

Significance:
Hundreds of thousands of Americans in rely on multi-weekly dialysis treatment to either supplement or supplant the function of their diseased kidneys. Dialysis and transplantation may remain the treatments of choice for End Stage Renal Disease for years to come, but patients suffering from acute and less-than-complete kidney failure might be able to benefit from the implantation of these tubule superstructures to aid in kidney function. Further research will need to address the viability of these grown tubules and analyze their metabolic activity, investigate the feasibility and logistics of implantation, and ultimately locate a source of cells and confirm a technique that will produce results appropriate for humans. With kidneys in high demand and low supply in the organ transplant market, any techniques that produce results to a similar end could improve length and quality of life for thousands of people across the globe.

Cell-patterning using poly (ethylene glycol)-modified magnetite nanoparticles.

Akiyama H, Ito A, Kawabe Y, Kamihira M, Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2009.


Summary: In tissue engineering, cell patterning is used to construct layers of different cell types in the goal of fabricating functional tissues and organs that similar to the native ones. In this article, cell patterning using PEG-Mags (polyethylene glycol- modified magnetic particles) and magnetic force is developed. A substrate surface can be chemically processed to control cell adhesion by varying hydrophilicity, while PEG-Mags region on the surface can resist the binding of cells. So when cells are seeded onto the surface, only the native region of substrate surface forms monolayer of cells. When PEG-Mags are added to a tissue dish, a 96-magnet plate will attract these magnetite particles into the pattern of magnets. Cells line are seeded and incubated for one day to form the designated pattern. Then the magnet will be removed and the dish will be washed with PBS to remove PEG-Mags and unattached cells. Another cell line can be seeded into the dish to grow a coculture of heterotypic cells.
Cell lines used in the experiment are mouse fibroblast NIH3T3, mouse myoblast C2C12 and immortalized human keratinocytes HaCaT. The stability of cell patterns is concerned since these cell lines are highly productive and fast in migration. This might become a problem if the dish is cultured for too long. Furthermore, the toxicity of the magnetite particles in cellular uptake is a main concern on cell viability. Experiment shows that PEG-Mags is safer choice than others such as aminosilane-Mags. The article suggested that PEG-Mags is very biocompatible and low toxic so they are employed for a novel cell-patterning method.
Significance: Cell-patterning has a very significant role in tissue engineering and cell biology so this simple and speedy method can be a very promising tool in the fields since recent methods in control cell adhesion by modifying surface hydrophilicity is time consuming and high cost. More researches will be conducted to such as cell patterning using primary cells to find the practical application of this novel method.

Using Embryonic Germ Cells To Engineer Adipose Tissue

Alexander T. Hillel, Shyni Varghese, Jennifer Petsche, Michael J. Shamblott, Jennifer H. Elisseeff. Embryonic Germ Cells Are Capable of Adipogenic Differentiation In Vitro and In Vivo. Tissue Engineering Part A. March 2009, 15(3): 479-486. doi:10.1089/ten.tea.2007.0352.

Summary:

Adipose tissue can be used as filler material for reconstructive and cosmetic surgeries such as breast reconstruction for mastectomy patients. However, transplantation of autologous adipose tissue is unfavorable for numerous reasons such as "poor revascularization, graft shrinkage, and volume loss, often resulting in a poor cosmetic outcome" (Hillel et al. 2009). It is therefore of interest to explore alternative methods of obtaining adipose cells and adipose tissue. Previous studies have shown that Mesenchymal Stem Cells (MSCs) can be successfully differentiated into adipose cells. However, there are several limitations to using MSCs to obtain adipose cells such as their limited in vitro proliferation and differentiation ability. The authors of this paper sought to show that embryonic germ cells can be differentiated into adipose cells as an alternative to MSCs.

Given the proper growth conditions, embryonic germ cells form embryoid bodies, which later yield embryoid-body derived (EBD) cells. EBD cells are pluripotent and have a greater capacity for long-term expansion, proliferation, and differentiation than MSCs. The purpose of this study is to examine whether EBD cells are capable of adipogenic differentiation in vitro and in vivo. In this study, human EBD cells were induced towards adipogenic differentiation (through exposure to adipogenic medium) in three different culture settings: (1) in vitro after being grown in a 2D monolayer, (2) in vitro after being encapsulated in a 3D poly(ethylene glycol) diacrylate (PEGDA) based hydrogel and (3) pre-differentiated in vitro after being encapsulated in a 3D PEGDA-based hydrogel and later implanted in vivo in four, 6 week old athymic-nude mice to test for the persistance of the adipogenic phenotype. Additionally, human MSCs were adipogenically differentiated using the same experimental conditions for each of the three different culture settings to allow for a direct comparison to EBD cells.

Results:

1. In vitro 2D monolayer culture

After being exposed to adipogenic medium for 21 days, both the EBD cells and MSCs tested positive for adipogenic differentiation by oil red-O staining, though the intensity of of the staining was greater in the EBD cells (Figure 1). However, the MSCs demonstrated a morphological change from a spindle shape to a spherical shape (which is more typical of adipose tissue) while the EBD cells retained a fibroblast-like spindle shaped morphology (Figure 1D, 1B). Reverse transcription PCR analysis also confirmed adipogenic differentiation of both EBD cells and MSCs.

Figure 1: in vitro 2D monolayer cultures after 3 weeks,
(A) 40x oil red-O staining of EBD cells, (B) 100x oil red-O staining of EBD cells,
(C) 40x oil red-O staining of MSCs, (D) 100x oil red-O staining of MSCs

2. In vitro 3D PEGDA hydrogel culture

After being exposed to adipogenic medium for 4 weeks, both the EBD cells and MSCs tested positive for adipogenic differentiation by oil red-O staining, and, simliar to what was observed in the 2D culture, the intensity of the staining was greater in the EBD cells (Figure 2). Both the differentiated EBD cells and differentiated MSCs were found to have a spherical morphology (characteristic of adipose cells) (Figure 2B, 2D). Gene expression of the adipogenic markers PPARγ, αP2, and LPL in the EBD cells and MSCs were comparable (Figure 3). In comparison to the 2D cultures, the differentiated EBD cells and MSCs grown in 3D cultures showed many-fold increases in gene expression of the aforementioned adipogenic markers, suggesting that cells differentiated in 3D culture are more representative of the cells in actual adipose tissue (Figure 4).

Figure 2: in vitro 3D hydrogel culture after 4 weeks of bathing in adiopogenic medium
(A) 100x oil red-O staining for lipids of EBD cells, (B) 400x oil red-O staining of EBD cells,
(C) 100x oil red-O staining of MSCs, (D) 400x oil red-O staining of MSCs

Figure 3: Reverse-transcription PCR results of in vitro 3D hydrogel cultures that demonstrates gene expression of several markers of adipogenic differentiation

Figure 4: Results of real-time PCR analysis that compares upregulation of several gene markers of adipogenic differentiation in undifferentiated (UD), 2D, and 3D in vitro cell cultures

3. In vivo culture

Four weeks after implantation, both the EBD cells and MSCs demonstrated strong oil red-O staining, and, similar to what was observed in the in vitro cultures, the intensity of the staining was greater in the EBD cells (Figure 5B, 5D). It was shown that as implantation time increased, LPL expression increased for both EBD cells and MSCs (Figure 5A, 5C). However, the EBD cells did not appear to express PPARγ and also showed decreasing expression of αP2 with time (αP2 disappeared altogether 3 weeks after implantation) (Figure 5A). In contrast, the implanted MSCs maintained expression of PPARγ, αP2, and LPL for the duration of 4 weeks after implantation (Figure 5C). The authors suggest that the EBD cells may have demonstrated decreased differentiation in the in vivo culture due to "a lack of signaling or poor host vascular invasion" (Hillel, et al. 2009).

Figure 5: in vivo culture
(A) Results of reverse-transcription PCR anaylsis that demonstrates gene expression of several markers of adipogenic differentiation for EBD cells and (C) for MSCs. (B) 400x Oil red-O staining for EBD cells and (D) for MSCs.

Significance:

The authors contend that "EBD cells demonstrated adipogenic differentiation comparable to that of MSCs" (Hillel et al. 2009). However, while the differentiated EBD cells maintain a stable adipogenic phenotype during in vitro growth, they appear to have difficulty maintaining their adipogenic phenotype once they are implanted in vivo. This indicates that adipogenically differentiated MSCs may be more suitable for clinical procedures that involve in vivo expansion.

Nonetheless, this study suggests two things: (1) In vitro adipogenic differentiation of EBD cells and MSCs grown in a 3D PEGDA-based hydrogel produces cells that more closely resemble actual adipocytes than EBD cells and MSCs that are differentiated in a 2D culture, and (2) EBD cells can indeed be used as an alternate source of adipocytes as they are able to proliferate and preserve a stable adipogenic phenotype when differentiated in a 3D PEGDA-based hydrogel structure. This is significant because it means that (1) if one is looking to engineer cells in vitro that closely resemble adipocytes, they may differentiate either MSCs or EBDs in a 3D PEGDA-based hydrogel structure, and (2) if one wishes to utilize some of the advantageous properties EBD cells have over MCSs (such as a greater proliferation rate after long-term expansion and the ability to differentiate after a greater number of population doublings) for procedures that involve engineering adipose cells in vitro, then they may because it was shown that EBD cells can differentiate to adipocytes in vitro with a degree of success that is comparable to MSCs. This study advances the field of adipose tissue engineering by helping to identify the ideal stem cell for specific situations (in vitro expansion or in vivo expansion).

IL-6 and Stat3 Are Required for Survival of Intestinal Epithelial Cells and Development of Colitis-Associated Cancer

Volume 15, Issue 3, 3 March 2009, Page 241,
Sergei Grivennikov, Eliad Karin, Janos Terzic, Daniel Mucida, Guann-Yi Yu, Sivakumar Vallabhapurapu, Jürgen Scheller, Stefan Rose-John, Hilde Cheroutre, Lars Eckmann, Michael Karin

Summary:

Colitis-associated cancer (CAC) is the major cause of death for ulcerative colitis patients around the world. Chronic inflammation release compounds that affect the growth and survival of cells. Previous studies have suggested that inflammation effects acts as tumor promoters. Immune cells produce cytokines and chemokines that propagate localize inflammatory responses that activate transcription factor, NF-kB. NF-kB enhance the growth and survival of premalignant cells. In cases of CAC, NF-kB-driven cytokine production by myeloid cells play an active role of CAC development . For intestinal epithelial cells (IEC), NF-kB activation produces cytokines that promote the survival and growth of premalignant cells created during the early phase of CAC tumorigensis.

In this paper, the identity of this cytokine produced by the NF-kB activation in myeloid cells is explored. Using CAC-induced mice, the ablation of the interleukin 6 (IL-6) cytokine produced by NF-kB was studied. This deficiency of IL-6 lowered the tumors in mice. The IL-6 decreased the tumor multiplicity in mice that suggests the role of IL-6 could be in early tumor promotion. The study examined the possible proliferation of intestinal epithelial cells (IECs) due to IL-6. IEC proliferation was enhanced by IL-6 as well as the apoptosis of IEC was lowered by IL-6 as well. With the regulation of IEC, the study continued to understand the cell source of IL-6 during tumorigenesis. Using bone marrow, the study results showed that Lamina propria and tumor-infilitrating CD11c+ (dendritic cells) CD11b+ (macrophages) are the major producers of IL-6 and then CD3+ (T cells) produce some IL-6 during CAC development. Within the same study, IL-6 was determined to be an important factor for tumor growth as well expression of genes required for premalignant cells during CAC tumorigenesis. The consequence of IL-6 deficiency resulted in the deficiency of phosphorylated Stat3. Stat3 is an activator of IECs during tumor growth. The absence of Stat3 in IEC disturbs the the tumor formation and growth of CAC. Thus, IL-6 major effect is the deficiency of Stat3. The cascade of NF-kB-IL-6-Stat3 is crucial to the development of tumor and growth of CAC.

The significance of this work is the identification of cytokines involved with tumor development and proliferation. Malignant cells survive through cytokines produced by inflammatory cells. Previous studies have shown that NF-κB activation in myeloid cells stimulate growth of premalignent intestinal epithelial cells (IEC) in colitis-associated cancer (CAC). This paper identifies NF-κB-dependent cytokine, IL-6 as the source for stimulus of premalignant IEC to survive and develop. The oncogenic transcription factor STAT3 mediates the effects of IL-6. Thus IL-6 and STAT3 could be targeted for future prevention and treatment of CAC