by: Harrison P. Nguyen, Peter L. Rady, Dr. Stephen K. Tyring

Abstract

Merkel Cell Carcinoma (MCC) is a highly aggressive skin malignancy hypothesized to affect Merkel Cells. Recently, the cancer was determined to have an infectious origin, a novel human DNA polyomavirus named Merkel Cell Polyomavirus (MCpyV). MCpyV expresses two non-structural proteins, the Large and Small T Antigens, which have been previously implicated in viral replication and survival. However, the Large T Antigen is found to harbor mutations prematurely truncating the MCpyV helicase and inhibiting its viral replication capability. Therefore, we propose the vital role of the Small T Antigen in the pathogenesis of the carcinoma. Here we review the hypothesized pathogenesis of the disease as well as the basic viral mechanism of the newly discovered virus.

Background

First discovered in 1875 by Friedrich Merkel, Merkel cells are present in the skin of all known vertebrates. Mice lacking the gene (Atoh1) necessary to produce Merkel cells fail to resolve fine spatial details,1 and thus, Merkel cells are hypothesized to function in the touch discrimination of shapes and textures. Recently, Merkel cells were shown to have an epidermal origin, rather than the previously hypothesized neural crest origin.2 In humans, Merkel cells are distributed along the basal zone of the epidermal, adnexal, and mucous membrane epithelium. Nevertheless, immunohistochemical analysis of Merkel cells shows staining for both epithelial and neuroendocrine markers. Merkel cells are oval-shaped, have an indented nucleus, and possess desmosomes that connect them with neighboring keratinocytes. In 1972, Cyril Toker first described an unusual, highly aggressive skin tumor with electron-dense neurosecretory granules. Since Merkel cells are the only cutaneous cells to form neurosecretory granules, the neuroendocrine carcinoma was attributed to the Merkel cells and subsequently named Merkel Cell Carcinoma (MCC). MCC is characterized by a painless, firm, red hemispherical tumor with a smooth, shiny surface that grows rapidly over a period of weeks to months.

Epidemiology

The American Cancer Society estimated 1,500 cases of MCC in the U.S. for 2008. This figure tripled the number of incidences reported twenty years ago, owing to improvements in methods of detection.3 Although the reported incidence remains ~65 times less than that of melanoma, MCC is twice as lethal; one in every three MCC patients will die from the malignancy.4 The mean age of the patients at time of diagnosis is ~70 years old.5 A strong link exists between MCC and ultraviolet light exposure. Regional incidence rates of MCC increase with sun exposure as measured by the UVB solar index.6 In particular, incidence of MCC is highest at equatorial latitudes. In one report, 81% of primary tumors occur on sun-exposed skin with 36% located on the face, the most sun-exposed anatomical site.7 Additionally, Caucasians have the greatest risk for developing the cancer. More notably, there is a clear association between MCC and immunosuppression. Chronically immunosuppressed individuals are 15 times morelikely to develop the malignancy than are age-matched controls. 4 For instance, MCC occurs more frequently in HIV and organ transplant patients (12/100,000/year) 5, and MCC is more lethal in immunosuppressed individuals with a mortality rate of up to 56%.7 Interestingly, there have been several documented cases of MCC regression following restoration of immune function. 8

Histology

Currently, the preliminary diagnosis is made on the basis of histopathology. The tumor has irregular margins, and its cells are arranged in strands or nests.9 Significant spacing between cells is typical, indicating a lack of cell-cell adhesion. As seen in many other cancers, the mitotic index in MCC tumors is very high, including many atypical mitoses.For definitive diagnoses, immunohistochemical analysis is usually required. Staining for cytokeratin 20 indicates a local aggregation of these filaments in a perinuclear dot-like pattern (Figure 1).

Pathogenesis

Currently, understanding of the molecular basis of MCC is still very limited. Because it is common in epithelial cancers, the well-described mitogen-activated kinase (MAPK) pathway has been extensively studied in MCC. As a result, it is clear how a mutation in this pathway can lead to transformation and especially immortalization. (MAPK traditionally plays a key role in many cell processes, including proliferation, suppression of apoptosis, migration, and differentiation;5 however, in several studies on MCC, little evidence has accumulated for a role of the MAPK pathway in tumorigenesis. Interestingly, traditional mutants in the MAPK pathway, such as the receptor tyrosine kinase c-kit and the ERK protein, which underlie other epithelial cancers are normal in MCC. 11,12 Although the MAPK pathway as a whole is generally inactive, a study demonstrated that inhibition of the farnesylation of the Ras protein, a particular component of the MAPK pathway, is sufficient to suppress MCC tumor growth in mice.13 The results of these studies taken together suggest the relevance of another Ras-regulated signal pathway involving the class 1 phosphoinositide 3 kinase (PI3K) and the Akt kinase. A downstream target of the PI3K/Akt pathway is the tumor suppressor p53. Induction of p53 accompanies apoptosis induced by Ras inhibition in MCC tumors.13 Although studies have indicated inconclusive evidence for the mutation of p53, the aforementioned finding that induction of p53 accompanies tumor suppression suggests that p53 expression and stability is related to MCC tumorigenesis, most likely through a downstream target of p53 in MCC progression. Protein Rb1 is a downstream target of p53 that functions as a key molecule in gene expression promoting the G1/S transition, and its demonstrated impact in virtually all cancers points to a significant role of Rb1 in MCC as well. In its hypophosphorylated state, Rb1 prevents cell cycle progression by inhibiting the E2F transcription factor. To enter S phase, cyclin-dependent kinases that are themselves indirectly regulated by p53 phosphorylate Rb and thus inactivate it, releasing E2F for cell cycle progression. Several mechanisms can allow Rb to mutate or become inactive, contributing to immortalization.5

Discovery of an infectious origin

With strong evidence supporting a correlation between immunosuppression and MCC, researchers long have hypothesized an infectious origin for the cancer. The mystery was partially unraveled in January 2008 when Feng et al. at the University of Pittsburgh identified a novel human DNA polyomavirus, which the authors named Merkel Cell Polyomavirus (MCpyV), in the full-genome sequencing of MCC samples. The authors developed a technique known as digital transcriptome subtraction (DTS), in which all mRNAs from a tumor are reverse transcribed into cDNA and then compared to the human genome. All human sequences are “subtracted,” and the remaining transcripts are assumed to be non-human sequences. Feng et al. conducted DTS with four MCC tumors, of which 99.4% of the sequences aligned closely with known human transcripts, and they established that one sequence common to all of the four tumors carried high sequence identity with the human BK polyomavirus antigen. Following extension and amplification, the authors identified the integration of a novel virus that has now been formally named MCpyV. To determine if MCpyV had a causal role, the researchers then screened ten MCC tumors for MCpyV using PCR. Seven were strongly positive and one was deemed weakly positive. Subsequent studies testing diverse populations have confirmed a ~80% presence rate of MCpyV DNA in MCC tumors, suggesting that MCpyV is a likely cause of MCC, although this has yet to be definitively confirmed.15, 16 Although all MCpyV tumors do not contain MCC, cancers that show presence of the virus are correlated with an increased potential for metastasis.17 Furthermore, based on Southern hybridization using a MCpyV DNA probe, Feng et al. determined that five of the eight MCpyV positive tumors contained monoclonal integration of the viral genome, suggesting that integration of the MCpyV genome occurs before metastases. In conjunction with the high frequency of MCpyV in MCC, this finding strongly indicates that MCpyV plays a causal role in tumorigenesis.

Polyomaviruses are small (40-50 nanometers in diameter), non-enveloped, circular, double-stranded DNA viruses. MCpyV is the sixth member of the Polyomaviridae genus that has been identified to infect humans. Polyomaviruses are highly species-specific and are thought to co-evolve with the organism that they infect. Their genomes are divided into three regions: early, late, and regulatory (Figure 2). The early region is expressed early in virus infection and continues during the late stage of infection. Importantly, the early region encodes non-structural proteins, namely the Large Tumor (T) Antigen and the Small Tumor (T) Antigen. The late region is expressed during and after genome replication and encodes structural proteins, in particular VP 1-3. The regulatory region contains transcriptional promoters and enhancers as well as the origin of replication. Because their genomes are rather basic, polyomaviruses rely on the host cell’s machinery for replication. The early-expressed genes bind to host proteins forcing the cell into S phase and thus facilitating viral replication. In previously discovered polyomaviruses, the Large T Antigen plays many diverse roles in viral replication. It is composed of three regions. Region 1 is essential in virion assembly, viral DNA replication, transcriptional control, and oncogenic transformation. Region 2 is important for host cell transformation as it contains an amino acid sequence that is important for binding the tumor suppressor pRB. Region 3 binds p53, most likely to promote cell growth and to facilitate host cell entry into S phase. In other polyomaviruses, the Large T Antigen plays an indispensable role in viral replication and survival. However, Shuda et al. identified MCC tumor-derived Large T Antigens to harbor mutations prematurely truncating the MCpyV LT helicase (22), which was confirmed by other publications (10) (Figure 3). The authors showed that with this truncation, the MCpyV Large T Antigen did not possess the capacity to replicate viral DNA but still preserved the ability to bind pRB. Thus, the MCpyV Large T Antigens is most likely still essential to the development of the carcinoma, but it does not contain the cell-lethal effects similar to the Large T Antigens of other polyomaviruses.8

Small T Antigen

With the interesting mutation and subsequent loss of function of the MCpyV Large T Antigen, questions arise as to how MCpyV can conquer host cells and replicate to infect more cells. Thus, the lab of Stephen Tyring at University of Texas at Houston Health Sciences Center proposes to focus on the other oncoprotein of interest, the Small T Antigen. In other polyomaviruses, the Small T Antigen plays a reduced, supplementary role to the Large T Antigen, but MCpyV could present a novel case where the Small T Antigen is the predominant pro-carcinogenetic component. The N-terminal sequence of the Small T Antigen is highly conserved among polyomaviruses, containing a heptapeptide region involved in cell replication. In addition, a J region (also present in the Large T Antigen) functions as a chaperone.18 However, the middle region of the Small T Antigen carries high variability, and consequently, its functionality in MCpyV is largely unknown. Most likely, it binds and inactivates protein phosphatase 2A (PP2A), an important biological enzyme for cells, and thus promotes S phase entry for the virus. In other polyomaviruses, Small T Antigen inhibition of PP2A is necessary for ST-mediated transformation to occur19(Figure 4).

PP2A refers to a family of serine-threonine phosphatases in eukaryotic cells. PP2A is composed of three subunits, each with several isoforms, allowing over 100 known PP2A complexes to exist. Particular PP2A complexes have characteristic substrates and functions within the cell, making it very difficult to establish the molecular mechanisms used by the Small T Antigen to induce transformation.20

The MCpyV Small T Antigen probably assumes a larger role in cell transformation and, in particular, replication, a process that was left unexplained given the mutation in the Large T Antigen. Thus, our lab sets out to elucidate the processes of the MCpyV Small T Antigen particularly binding partners and its behavior in a host cell environment. We already have shown that the Small T Antigen is highly conserved among MCC tumors containing the MCpyV genome, and we hope that information regarding the behavior of the protein will answer important questions regarding the pathogenesis of Merkel Cell Carcinoma.

The proposed work is a stepping stone in the struggle to understand and cure cancer. Although its prevalence is dwarfed by its melanoma and non-melanoma counterparts, Merkel Cell Carcinoma is rapidly growing in incidence rates and its 33% fatality rate presents a rather bleak outlook for the unfortunate individuals who are diagnosed with the disease. This research offers hope for understanding the mechanism by which the viral origin is able to transform and immortalize cell targets, subsequently leading to rapid metastases. The implications of this research are farreaching; knowledge into the mechanism of infection and viral survival is expected to shed light on questions relating to virology, Merkel cell function, and cancer in general.

Harrison Nguyen is a sophomore Cognitive Sciences major at Hanszen College. He is primarily interested in infectious carcinogenesis, and he works in the mucocutaneous laboratory at University of Texas Health Sciences Center under the guidance of Dr. Stephen Tyring. His laboratory is specifically studying the role of the Small Tumor Antigen in Merkel Cell Carcinogenesis. Harrison aspires to pursue his passion of medicine in the field of dermatological oncology as a research-physician.

References

1. Maricich SM, Wellnitz SA, Nelson AM, Lesniak DR, Gerling GJ, Lumpkin EA, Zoghbi HY. 2009. Merkel Cells Are Essential for Light-Touch Responses. Science. 324: 1580 – 1582.
2. Morrison KM, Miesegaes GR, Lumpkin EA, Maricich SM. 2009. Mammalian Merkel cells are descended from the epidermal lineage. Dev Biol. 336(1):76-83.
3. Lemos B, Nghiem P. 2007. Merkel cell carcinoma: more deaths but still no pathway to blame. J. Invest. Dermatology. 127: 2100-2103.
4. Heath M, Jaimes N, Lemos B, Mostaghimi A, Wang LC, Penas PF, Nghiem P. 2008. Clinical characteristics of Merkel cell carcinoma at diagnosis in 195 patients: the AEIOU features. J. Am. Acad. Dermatol. 58: 375-381.
5. Becker JC, Schrama D, Houben R. 2008. Merkel cell carcinoma. Cell. Mol. Life Sci.
6. Agelli M, Clegg LX. 2003. Epidemiology of primary Merkel cell carcinoma in the United States. J. Am. Acad. Dermatology. 49: 832-841.
7. Penn I, First MR. 1999. Merkel’s cell carcinoma in organ recipients: report of 41 cases. Transplantation. 68: 1717-1721.
8. Garneski KM, DeCaprio JA, Nghiem P. 2008a. Does a new polyomavirus contribute to Merkel cell carcinoma? Genome Biology. 9: 228.
9. Plaza JA and Suster S. 2006.  The Toker tumor: spectrum of morphologic features in primary neuroendocrine carcinomas of the skin (Merkel Cell Carcinoma). Ann. Diagn. Pathol. 10:376-385.
10. Sastre-Garau X, Peter M, Avril MF, Laude H, Couturier J, Rozenberg F, Almeida A, Boitier F, Carlotti A, Couturaud B, Dupin N. 2009. Merkel cell carcinoma of the skin: pathological and molecular evidence for a causative role of MCV in oncogenesis.  J. Pathol. 218(1):48-56
11. Strong S, Shalders K, Carr R, Snead DR. 2004. KIT receptor (CD117) expression in Merkel Cell Carcinoma.  Br. J. Dermatol. 150: 384-385.
12. Houben R, Michel B, Vetter-Kauczok CS, Pfohler C, Laetsch B, Wolter MD, Leonard JH, Trefzer U, Ugurel S, Schrama D, Becker JC. 2006. Absence of classical MAP kinase pathway signaling in Merkel cell carcinoma. J. Invest. Dermatol. 126: 1135-1142.
13. Jansen B, Heere-Ress E, Schlagbauer-Wadl H, Halaschek-Weiner J, Waltering S, Moll I, Pehamberger H, Marciano D, Kloog Y, Wolff K. 1999. Farnesylthiosalicylic acid inhibits the growth of human Merkel cell carcinoma in SCID mice. J. Mol. Med. 77: 792-797.
14. Feng H, Shuda M, Chang Y, Moore PS. 2008. Clonal Integration of a polyomavirus in human Merkel Cell Carcinoma. Science. 319: 1096-1100.
15. Kassem A, Technau K, Kurz AK, Pantulu D, Loning M, Kayser G, Stickeler E, Weyers W, Diaz C, Werner M, Nashan D, zur Hausen A. 2009. Merkel cell polyomavirus sequences are frequently detected in nonmelanoma skin cancer of immunosuppressed patients. Int. J. Cancer. 000: 000-000.
16. Duncavage EJ, Zehnbauer BA, Pfeifer JD. 2009. Prevalence of Merkel cell polyomavirus in Merkel cell carcinoma. Modern Pathology. 1-6.
17. Garneski KM, Warcola AH, Feng Q, Kiviat NB, Leonard JH, Nghiem P. 2008. Merkel Cell Polyomavirus is More Frequently Preesent in North American than Australian Merkel Cell Carcinoma Tumors. Journal of Investigative Dermatology.
18. Khalili K, Sariyer IK, Safak, M. 2008. Small Tumor Antigen of Polyomaviruses: Role in Viral Life Cycle and Cell Transformation.  J. Cell. Physiol. 215: 309-319.
19. Hahn WC, Dessain SK, Brooks MW, King JE, Elenbaas B, Sabatini DM. 2002. Enumeration of the SV40 early region elements necessary for human cell transformation. Molecular and Cellular Biology. 22(7): 2111-2123.
20. Sablina AA, Hahn WC. 2008. SV0 small T antigen and PP2A phosphatase in cell transformation. Cancer Metastasis Rev. 27: 137-146.
21. Miller RW, Rabkin CS. 1999. Merkel Cell Carcinoma and Melanoma: Etiological Similarities and Differences. Cancer Epidemioology, Biomarkers & Prevention. 8: 153-158.
22. Shuda M, Feng H, Kwun HJ, Rosen ST, Gjoerup O, Moore PS, Chang Y. 2008. T Antigen mutations are a human tumor-specific signature for Merkel cell polyomavirus. PNAS. 105: 16272-16277.

by: Deepika Satish

Abstract

Studying the evolution of globins and observing patterns of oxygen migration is one method of determining how nature has selectively chosen properties for oxygen storage and transport. In this project, we have studied Scapharca inaequivalvis Hemoglobin I (ScHbI), a dimeric protein found in Scapharca inaequivalvis clams, to map its internal migration of oxygen and compare the observed pathway to the distal histidine ‘gate’ pathway found in both monomeric mammalian myoglobins and tetrameric adult human hemoglobin. For ScHbI, this putative distal histidine gate (His-69(E7)) appears to be closed by the hemoglobin’s inverted quaternary structure; thus, we tested if it was the ligand migration pathway in ScHbI by first changing the size and polarity at and around the distal histidine gate and then examining the effects on oxygen association, dissociation, and affinity. Our results strongly suggest that, despite facing the dimer interface, ScHbI’s E7 residue functions as the gate for diatomic ligand capture in the distal pocket, thus having a similar oxygen migration pathway as mammalian myoglobins and human hemoglobins. By studying ScHbI and other model hemoglobins, we are building up a set of rules that govern the function of all hemoglobins. These rules can then be employed to optimize the properties of recombinant human hemoglobin and to engineer a hemoglobin-based oxygen carrier or blood substitute for the treatment of hemorrhagic shock due to severe blood loss.

Blood Substitutes

As the demand for whole donated blood increases every day, the need for blood substitutes becomes more and more important for the treatment of hemorrhagic shock under emergency situations. Over the years, much effort has been expended on making safe, effective blood substitutes, but none have been able to perfectly emulate human hemoglobin (HbA) within intact red blood cells. A hemoglobinbased oxygen carrier (HBOC) named PolyHeme® is one of the few oxygen-carrying blood substitutes to complete a phase III trial. However, it failed to receive FDA approval in April 2009 because the studies revealed that “Polyheme® places the patients at a higher risk of significant adverse events”,1 such as heart attacks and myocardial infarctions.2 Another hemoglobin-based blood substitute known as Hemopure ® is the first and only oxygen-carrying blood substitute to be approved for human use in South Africa. However, in the United States, analyses of the phase III human trials conducted using Hemopure® were brought into question, and in mid-2002, the FDA mandated that further animal trials on nitric oxide scavenging and blood pressure effects be conducted before any new human clinical trials could resume.3,4 This brings to view just one of the many obstacles faced when developing blood substitutes. When hemoglobin- based blood substitutes have low oxygen association, they tend to bind nitric oxide (known as NO scavenging), which triggers vasoconstriction. This results in hypertension, a major adverse effect of current hemoglobin-based blood substitutes.

Dr. John S. Olson’s laboratory has focused on engineering recombinant hemoglobin (Hb) that has fewer blood pressure side effects and increased oxygen delivery efficiency. His laboratory has conducted multiple molecular biophysics studies exploring the optimization of the properties required for a successful blood substitute. Furthermore, recombinant hemoglobins from multiple organisms are being studied to understand how their unique structures affect their oxygen binding properties and to apply this knowledge in developing strategies for engineering extracellular hemoglobin as a blood substitute.

Engineering Recombinant Hemoglobin

Designing recombinant hemoglobin for use as a blood substitute necessitates the creation of a mutant with moderately low oxygen affinity, large oxygen exchange rates, high subunit cooperativity, and minimum rates of nitric oxide scavenging.5 In order to synthetically optimize the function of recombinant hemoglobin in attaining these properties, we require a general understanding of how globins evolved in nature under selective pressure for cooperative and rapid oxygen binding and release. A general knowledge base will enable us to develop a set of rules governing the speed of ligand (oxygen and carbon monoxide) migration into the protein and the stereochemistry of iron-ligand bond formation within the globin active site. With a progressively better understanding of oxygen migration paths within these proteins, we will be able to understand how gases migrate within proteins in general and to identify patterns of pathways for different families of hemoglobins.6 Most importantly, we will be able to predict oxygen pathways in various types of hemoglobin and modify them by protein engineering. Although many different types of oxygen pathways in globins have been mapped thus far, the best understood is sperm whale myoglobin (SwMb). Its oxygen interactions have been studied extensively, and it has served as the model system of protein dynamics for many years. 7,8 While SwMb studies have helped shed light on the basics of oxygen migration, a key feature of the globins that has not been extensively explored is if or how cooperative interactions between subunits alter ligand pathways. Multimers, such as HbA, often display cooperativity through inter-monomer conformational changes. 9,10 In this study, hemoglobin from the mollusk Scapharca inaequivalvis (ScHbI) was chosen as the model system because it represents the simplest allosteric system of two identical subunits in which one subunit alters the other’s ligand affinity at a chemically identical active site and because the interface appears to block the distal histidine gate found in mammalian Mbs and Hbs. 11

Scapharca inaequivalvis Hb I

Mammalian hemoglobin is a complex tetrameric protein that delivers oxygen to and transports carbon dioxide away from various tissues in the body (Figure 1A). The protein has four subunits, each containing a prosthetic group known as heme. Each heme molecule contains one iron that binds to a single diatomic oxygen molecule. In SwMb and HbA, ligands gain access to this heme located in the ligand binding pocket by moving through a channel created by upward and outward movement of the distal histidine E7 side chain. This mechanism for ligand entry is known as the His(E7) gate. ScHbI is thought to have a His(E7) gate similar to that in Mb and HbA.8,12 However, the His(E7) gates of this dimeric hemoglobin appear to be closed by its inverted quaternary structure, as each subunit seems to cover and block the outward movement its adjacent subunit’s His(E7) gate. Thus, to bind to the heme within the distal pocket, oxygen would have to first migrate into the dimer interface, and then proceed past the His(E7) gate (Figure 1B),13 unlike in HbA, which has ‘open’ (E7) gates that are not blocked by the molecule’s own subunits (Figure 1A). Thus, whether oxygen still enters and exits ScHbI through the E7 gate is an open question.7

In this project, mutagenesis has been conducted at five key helical positions (E7, CD3, CD4, F4, and B3), which are either at the E7 gate or within the ligand pocket, to create the library of ScHbI mutants, most of which are shown in Figure 3. These mutations were intended to demonstrate a wide range of effects, all of which were chosen to determine if the E7 gate has a role in ligand migration. If oxygen enters through the E7 gate, then varying the size and polarity of the amino acids at the putative gate (E7) should show large effects on the rate of ligand association; altering the ease of opening of the gate and the amount of internal water that must be displaced before oxygen can bind would result in measureable changes in oxygen association and dissociation.14 Altering amino acids within the distal pocket (at positions CD3 and CD4) should affect the ease of movement of the His(E7) gate and thus the rate of migration of ligands into and out of the distal pocket. Lastly, mutations to reduce cooperativity (at position F4) and to render a monomeric form of the protein (at position B3) should simplify the model such that the role of the E7 gate can be further elucidated.15,16,17 The recombinant hemoglobin protein was constructed based on the protocol prescribed by Summerford et al.18 By observing the effects of these changes in the oxygen binding rate and equilibrium constants of the hemoglobin and identifying the trends that depend on the size and flexibility of the side chain at the E7 position, we can determine whether the distal histidine gate is the route of ligand migration in ScHbI.

Mapping Ligand Migration

Time courses for O2 and CO binding were measured and used to determine ligand association (k’) and dissociation (k) rates and equilibrium association constants (K). By looking at trends in the oxygen rate and affinity constants for the library of ScHbI mutants in terms of the size and polarity of the altered side chains, we can map the pathway of oxygen migration and examine the strength of electrostatic and steric interactions with the bound ligand.

In these kinetic experiments, CO is photodissociated from the heme group of ScHbI in the presence of O2 by a flash tube-driven dye laser, which provides an intense light pulse for ~0.5 μs at 577 nm. When the absorbance changes in the sample are followed at 436 nm, we can observe the formation of deoxyHb (hemoglobin without any bound ligand) during the laser excitation pulse, and then follow its decay as ligands rebind from solvent in a bimolecular process. The initial fast bimolecular rebinding phase (kf) represents rebinding of O2 and some CO at the rate given in the equations below. If the absorbance changes are followed at 425 nm, a slow unimolecular phase (ks) is observed and represents the displacement of oxygen that was bound in the fast phase by carbon monoxide, which has a greater affinity for the protein. Expressions for the rate of this slow phase and the overall oxygen affinity constant, which is calculated from the association (k’O2) and dissociation (kO2) constants, are also given below:

Sets of k’O2, kO2, and KO2 were obtained for the different mutants, after which trends and correlations with increasing size or polarity were examined.

Analysis of the kinetic data for ScHbI suggests that the major pathway for ligand entry and exit in ScHbI is through the E7 gate despite the gate’s apparent closure by the adjacent subunit. In general, increasing the size of the amino acid at the E7 position decreases the rate of oxygen association up to 10-fold, indicating the involvement of the E7 gate in oxygen migration. This is further supported by the fact that changing the polarity of the E7 amino acid has a great effect on the rates of both ligand association and dissociation. Polar amino acids that are capable of donating a hydrogen bond markedly stabilize bound oxygen. However, these same amino acids stabilize water in the binding pocket of deoxyScHbI and slow the rate of ligand association. Thus, when histidine, asparagine, and glutamine are present at the E7 position, there is a complex tradeoff between the effects of size and polarity of the amino acid on oxygen association and dissociation rates. Mutations at the CD4, CD3, F4, and B3 positions also indicate that the main path of ligand migration is through the dimer interface and the E7 gate.

In general, the pattern of mutagenesis effects observed for ScHbI resembles those reported previously for the subunits of HbA and SwMb, which suggests that the distal histidine moves outward to allow diatomic ligand entry and exit.12,8. Thus, our work with ScHbI indicates that the basic rules governing the ligand migration and affinity in ScHbI are identical to those for mammalian Hbs and Mbs and apply in general to all globins with a distal histidine.

Future Work

While the experiments done so far on ScHbI all indicate that the E7 gate is the route of ligand migration, a few more mutants need to be made to fully demonstrate the range of effects that the mutations have on the rate of oxygen association and dissociation. This project has enabled us to further understand the evolution of globins and helped us to elucidate a model on which future experiments can be based, but much work still needs to be done to completely understand animal hemoglobins and establish principles for globin design. Properties required for a hemoglobin-based blood substitute include moderately low oxygen affinity, high oxygen exchange rates, high subunit cooperativity, discrimination against CO binding, low rates of nitrogen oxide reaction, resistance to autooxidation, resistance to reactions of H2O2, resistance to denaturation, and high affinity to heme,5 of which only the first three properties were studied following the methods used in this project. However, all of these key ideas must undergo more extensive investigation, especially in HbA. Continuing research of hemoglobins of other organisms is also crucial to understanding the patterns for all of these properties throughout evolution.

Deepika Satish has worked in Dr. John S. Olson’s lab where she has constructed recombinant ScHbI, performed kinetic tests on the recombinant protein, and analyzed the trends of the kinetic tests in relation to the HbA. Previous such hemoglobin research conducted in the Olson lab has yielded ideas that have led to the construction of safer second-generation hemoglobin-based blood substitutes by the Baxter Hemoglobin Therapeutics.5 Continued research in this area will hopefully contribute more elucidations and ideas for more efficient and effective blood substitute construction. Deepika is a sophomore Biochemistry and Cell Biology major at Will Rice College ’12.

by: Navonil Ghosh

Abstract

Angiogenesis, the growth of new blood vessels from preexisting vasculature, is a significant physiological process in living organisms. Vascular endothelial growth factors, or VEGF, and associated receptors are key effectors during the formation of blood vessels. Because any defects or abnormalities in vessel formation can result in a variety of vascular diseases and disorders, an intimate understanding of the developmental pathways that regulate angiogenesis could elucidate the causes of these diseases and reveal possible treatments or preventative measures. Thus, scientists are exploring a number of angiogenic signaling pathways and effectors of these pathways, particularly VEGF. By manipulating these pathways and analyzing the resulting phenotypic effects on blood vessel formation, researchers can determine the effectiveness of angiogenic regulatory molecules as therapeutic agents for vascular disorders.

Introduction

The vasculature of vertebrate organisms is regulated by a number of signaling pathways that affect vascular endothelial cells, which comprise the inner lining of blood vessels. These pathways govern both the growth and patterning of blood vessel formation. Any disruption to the developmental cues necessary for proper growth can result in defective vascular formation that can lead to a number of diseases including psoriasis, rheumatoid arthritis, and cancer. Because the blood vessels of the vascular system are intricately linked to the survival of all vertebrates, a vast number of clinical and developmental studies have focused on angiogenesis.

Although the term was first used by Dr. John Hunter in 1787 to describe blood vessel growth in reindeer antlers, angiogenesis entails the formation of new capillary blood vessels in all vertebrates and is a major area of developmental study.1 In healthy vertebrate organisms, angiogenesis is a key component to healing wounds and restoring circulation to damaged tissue. The process is controlled in a healthy organism by a series of angiogenesis-stimulating growth factors and angiogenesis inhibitors, which serve as the “on” and “off” switches for blood vessel growth.2 The angiogenic process was thus proposed to have a therapeutic role by means of new vessel induction in damaged tissue or inhibition of vascular formation in pathological pathways involving abnormal vessel growth. According to the Angiogenesis Foundation, approximately 500 million patients in Western nations alone could benefit from either proangiogenic or antiangiogenic therapy. As a result, over $4 billion in medical research has been devoted to developing angiogenic-based therapeutic drugs.1

Angiogenic Vessel Formation

Angiogenesis is dependent on a signaling cascade to induce vascular development (Fig. 1).1 The first step of the angiogenic process is the release of angiogenic growth factors that bind to the membrane-bound receptors of nearby vascular endothelial cells. These activated endothelial cells then start producing and secreting enzymes that degrade the basement membrane, a sheath-like covering of blood vessels. They also initiate an intracellular signaling cascade that prompts the formation of other building blocks of endothelial cell formation. The endothelial cells divide and pass through the dissolved openings of the basement membrane to where the new vessel will form. The sprouting of the newly developing vessel is aided by adhesion molecules known as integrins that bind and help pull the vessel into place. Meanwhile, other enzymes such as matrix metalloproteinases degrade the surrounding extracellular matrix to make room for the growing vessel. The endothelial cells then join together to form individual capillary sprouts, which are stabilized by smooth muscle cells and pericyte muscle cells. Blood circulation begins when the individual tubes join together to form blood vessel loops.

Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is one of the most critical effectors involved in angiogenesis. VEGF, a family of tyrosine kinases, stimulates a variety of signaling pathways that regulate the growth of endothelial cells.3 The VEGF system involves a series of ligands and receptors. The secreted ligands include VEGF-A, VEGF-B, VEGF-C, VEGF-D and PlGF and the receptors include VEGF-R1, VEGF-R2, VEGF-R3. Through autophosphorylation by the tyrosine kinases of the VEGF family, the VEGF receptors activate cellular signaling pathways that ultimately control migration, survival, and proliferation of vascular endothelial cells. Because VEGF plays such an intimate role in angiogenesis, it is highly utilized in a variety of scientific studies involving other angiogenic factors.4 A recent study, for example, shows that angiomodulin, a modulator of VEGF-dependent vascular formation, has increased expression in human tumor tissue.5 Although further examination of angiomodulin’s role in cancer progression is needed, this could mark a step in evaluating angiogenic pathways in a therapeutic role for cancer.

The Zebrafish Model

There are a variety of in vivo models available for angiogenesis (Table 1).6 The zebrafish model organism, Danio rerio, is particularly well-suited for the analysis of developing vasculature structures for a variety of reasons. It produces a large clutch size of transparent, external embryos that allow easy, accessible observation from an early age.7 Furthermore, the rapid development of zebrafish embryos also allows for timely analysis and easy reproduction of experimental results. The genetic pathways of zebrafish share many angiogenic mechanisms and genes with other vertebrates, and because these pathways can be easily manipulated, the zebrafish can serve as a useful tool for analyzing the development of vasculature in vertebrate organisms in general.8 Moreover, the vasculature itself can be easily observed through the use of transgenics, allowing the intersegmental, subintestinal, and cranial vessels of the organism to be easily examined (Fig 2.).9 By virtue of these advantages, the zebrafish is a highly used model organism by scientists studying the processes that regulate blood vessel formation.

In order to reproduce defects in the vasculature of the zebrafish model organism, scientists rely on a number of effective assays and experimental procedures. In order to visibly examine the blood vasculature of zebrafish, transgenic zebrafish with fluorescent vessels have been developed. Fluorescent expression through the insertion of the green fluorescent protein (GFP) construct into fli-eGFP, mTie2-GFP, and flk-GFP promoters, which are expressed in vascular endothelial cells, allows the entire vasculature of the organism to be visible.8 Microinjection of mRNA and morpholinos can be used to examine a target gene’s role in vascular development by showing the effects of altering normal gene expression.7 Injecting mRNA overexpresses the target gene, while the morpholinos block translation to knockdown the gene. Injections into transgenic zebrafish allow the effects of overexpression and knockdown to be examined in great detail. Further genetic analysis can be done through in situ hybridizations, which uses antibodies to stain a complementary strand of RNA that binds to a target gene. This allows for the identification of changes in the expression of other genes through alteration of the original target gene.

Angiogenic-Dependent Diseases

Angiogenesis-based pathological conditions can occur when the body loses control over the angiogenic process and blood vessel formation progresses either excessively or insufficiently .1 In diseases dependent on excessive angiogenesis, diseased cells release excessive amounts of angiogenic growth factors that cause abnormal proliferation of the endothelial cells that make up newly developing vessels. These excessively sprouting blood vessels damage healthy tissue while continuing to nourish the diseased tissue. Conditions related to an overgrowth of blood vessels include macular degeneration, diabetic blindness, and psoriasis. Abnormal vessels can even allow cancerous growths to metastasize by providing the means for tumor cells to reach healthy organs. In the case of insufficient angiogenic vessel growth, the body cannot produce enough angiogenic growth factors for proper vessel formation. The lack of adequate blood vessel growth affects tissue perfusion and can cause necrosis. Insufficient angiogenesis is involved with both coronary artery disease and stroke.

Angiogenic Therapy and Inhibition

Because so many pathological conditions have a tie to abnormal blood vessel formation, scientists have proposed that angiogenesis may have therapeutic purposes. Therapeutic angiogenesis generally involves turning the genes controlling the process, or using angiogenesis – stimulating growth factors to allow for the growth of new blood vessels.1 Angiogenic stimulation targets vascular endothelial cells by using cytokine growth factors, such as those of the VEGF family. The resulting increase in endothelial cell proliferation enhances vessel formation and blood profusion to damaged, or ischemic, tissue. This proangiogenic stimulation is induced in order to enhance blood flow to injured tissue, transport survival factors to areas in need of tissue repair, activate populations of regenerative stem cells, and ultimately restore tissue to its normal state.

A number of clinical drugs and interventions have been developed to treat pathologies through therapeutic angiogenesis. The three diseases most commonly treated using angiogenic therapy are chronic wounds, such as arterial or venous ulcers, peripheral arterial disease, and ischemic heart disease.1 To treat these diseases, angiogenesis can be stimulated through therapeutic angiogenic drugs. An FDA-approved growth-factor based drug known as rhPDGF is a recombinant protein medication used to treat diabetic ulcers. Systems such as autologel or SmartPReP allow for the treatment of injured tissue using autologous blood samples, which contain the natural growth factors and cytokines needed to stimulate healing through angiogensis. 10, 11 Early clinical trials of other angiogenic stimulators have shown evidence of increased blood flow and function of the left ventricle. This is a promising therapeutic tool for treating coronary heart disease. Angiogenesis can also be promoted without relying on drugs or medication. Negative pressure wound therapy stimulates blood vessel growth through micro-deformations of tissue that respond with signal transduction leading to epithelial proliferation. Other methods include the MIST ultrasound, a low-frequency and low-intensity procedure that stimulates endothelial cells and enhances blood perfusion, and Hyperbaric Oxygen (HBO), which increases expression of VEGF and endothelial progenitor cells.12, 1

Inhibition of angiogenesis can also have therapeutic properties, for it can prevent feeding of diseased tissue such as cancerous tumors. Anti-angiogenic agents inhibit cell signaling pathways necessary for vessel growth. These inhibitors include three major varieties: monoclonal antibodies, tyrosine kinase inhibitors, and inhibitors of mammalian targets of rapamycin (mTOR).1 Monoclonal antibodies bind to VEGF to prevent it from binding in turn to VEGF receptors, thus interrupting the proliferation of endothelial cells. Small molecule tyrosine kinase inhibitors interrupt pathways involving a number of tyrosine kinase growth factor receptors, including VEGF receptors. mTOR inhibitors intercept the PI3/AKT pathway necessary for tumor proliferation. 13 All three methods act by inhibiting a pathway necessary for endothelial proliferation and thus prevent angiogenic nourishment of diseased or cancerous tissue.

Conclusions

Therapeutic proangiogenic and antiangiogenic applications have shown potential in alleviating a number of vascular-based pathologies. The zebrafish developmental model remains a valuable tool for analyzing vascular formation and potential angiogenic therapies. As our understanding of angiogenesis increases, advancements in therapeutic uses for the process will have an important impact on clinical treatment of vascular pathologies.

Acknowledgements

I would like to thank Dr. Daniel Wagner for introducing me to the angiogenic and zebrafish research. I would also like to thank Jane Li, Casey O’Grady, Eric Kim, and Dr. Amina Qutub for their feedback and advice upon editing my article.

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Navonil Ghosh is a sophomore from Hanszen College majoring in Biochemistry and Cell Biology. Under the supervision of Dr. Daniel Wagner of the Biochemistry and Cell Biology Department at Rice University, Ghosh is investigating the role of the aggf1 gene in angiogenesis. Angiogenic Factor with G patch and FHA domains 1 (AGGF1) has been associated with the VEGF pathway, as well as a vascular birth defect known as Klippel-Trenaunay-Weber Syndrome. Thus, aggf1 is hypothesized as an important regulator of vascularization in zebrafish. Ghosh is analyzing the effects of overexpression and knockdown of aggf1 on the vasculature of the zebrafish model in order to better understand the role of aggf1 in angiogenesis and evaluate its therapeutic potential.