Specific Aims
Vascular Ehlers Danlos Syndrome (vEDS) is a subtype of Ehlers Danlos Syndrome. Ehlers Danlos Syndrome (EDS) is an umbrella term for the group of 13 different connective tissue disorders often affecting collagen function. [1]. In vEDS, type 3 collagen is affected which is important for the lining of blood vessel walls. Major complications of the disorder include tearing, rupture, and dissection of the blood vessel, uterine, and intestinal walls [2]. Amino acid substitutions in the COL3A1 gene (critical for collagen type 3 synthesis and tensile strength) result in vEDS. Some studies report median patient survival rates to be lower among males compared to females. Additionally, in males there is a higher incidence of lethal major complications by the age of 20. The biological reasons for this are still unclear.
My objective is to study the molecular basis for sex differences in vEDS. To do this I will use Zebrafish which are excellent model organisms for vascular visualization. While Zebrafish do not make the COL3A1 protein, their COL1A1 protein contains the same protein domains and similar functionality to COL3A1. The long-term goal is to better understand the genetic causes for sex differences in presentation of vascular complications in Vascular Ehlers Danlos Syndrome for future treatment and potentially prevention of major complications. I hypothesize that male mutant juvenile Zebrafish will experience increased vascular complications at an earlier age due to differential gene expression levels and reduced presence of integrin and collagen binding protein interactions.
Aim 1: Identify conserved amino acid sites critical for vEDS vascular events in males and females
Approach 1: An NCBI BLAST of COL3A1 in humans and COL1A1 in Zebrafish will be conducted and the FASTA sequences retrieved and aligned by Clustal Omega software. A conserved single amino acid residue within the collagen triple helix repeat protein domain will be identified for mutation. Using Base Editing, the identified point-mutation will be generated in a Zebrafish model. Male and female wildtype Zebrafish, COL1A1 mutant Zebrafish with a known vEDS phenotype (COL1A1 c.934C>T) [3], and the newly identified mutation will be used as the study arms. The vasculature of each organism will be visualized and measured in each sex group for signs of aneurysms and vascular abnormalities. The Hypothesis is that mutations in the alpha triple-helix region will result in a dysfunctional COL1A1 protein leading to weakened vasculature. The Rationale is that the COL1A1 homolog in Zebrafish will be an effective model as it contains the same protein domains relating to fibril collagen synthesis in vEDS.
Aim 2: Examine sex-differences in differential gene expression for disease vascular phenotypes
Approach 2: RNA-seq will be conducted on aortic tissue for both wild type male and female zebrafish and those harboring the COL1A1 vEDS mutation. This will be done in juvenile Zebrafish developmentally as that is when presentation of vEDS sex differences are the highest. The gene expression profiles can be stratified by sex and sorted by integrin and collagen binding GO terms. Genes that are differentially expressed between sexes and wildtype/disease Zebrafish could indicate different molecular mechanisms in disease. These genes could then be induced in Zebrafish using a CRISPR/Cas9 system to study disease phenotype. The Hypothesis is that there will be differential gene expression in aortic vascular wall tissue relating to integrin and collagen binding between male and female zebrafish with vEDS. The Rationale is that males and females present with different disease phenotypes at an early age, and RNA-seq will allow for gene expression differences within aortic wall tissues to be analyzed.
Aim 3: Examine sex-differences in integrin and collagen binding proteins for vEDS
Approach 3: Integrins are critical for ECM cell-wall adhesion and attach to the subendothelial ECM when a vascular injury occurs [4]. Each of the male and female mutant zebrafish, along with wild type controls, will have aortic tissue harvested to examine the presence of integrins and collagen binding proteins. The aortic tissues would be chemically tagged in an iTRAQ system, and the proteins detected through mass spectrometry. For functional analysis, the proteins present will be sorted by GO terms regarding ECM and integrins, which are expected to be less enriched in males compared to females. Knockouts of relevant genes will be completed in male zebrafish to confirm functionality. The Hypothesis is that males will have a decreased presence of collagen binding proteins and integrins which help protect against injury during vasculature rupturing. The Rationale is that integrins bind to motifs on the collagen sequence in COL1A1, and vEDS mutations have been shown to hinder integrin binding ability and functionality.
References
[1] The Ehlers-Danlos Society. (2017). What are the Ehlers-Danlos Syndromes? | The Ehlers Danlos Society. The Ehlers Danlos Society. https://www.ehlers-danlos.com/what-is-eds/
[2] Byers PH. Vascular Ehlers-Danlos Syndrome. 1999 Sep 2 [Updated 2019 Feb 21]. In: Adam MP, Feldman J, Mirzaa GM, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2024. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1494/
[3] Cortini, F., Marinelli, B., Romi, S., Seresini, A., Pesatori, A.C., Seia, M., Montano, N., & Bassotti, A. (2017). A New COL3A1 Mutation in Ehlers-Danlos Syndrome Vascular Type With Different Phenotypes in the Same Family. Vascular and Endovascular Surgery, 51, 141 - 145.
[4] Hoop, Cody L. et al., Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers–Danlos syndrome–associated substitutions in Journal of Biological Chemistry, Volume 294, Issue 39, 14442 - 14453
My objective is to study the molecular basis for sex differences in vEDS. To do this I will use Zebrafish which are excellent model organisms for vascular visualization. While Zebrafish do not make the COL3A1 protein, their COL1A1 protein contains the same protein domains and similar functionality to COL3A1. The long-term goal is to better understand the genetic causes for sex differences in presentation of vascular complications in Vascular Ehlers Danlos Syndrome for future treatment and potentially prevention of major complications. I hypothesize that male mutant juvenile Zebrafish will experience increased vascular complications at an earlier age due to differential gene expression levels and reduced presence of integrin and collagen binding protein interactions.
Aim 1: Identify conserved amino acid sites critical for vEDS vascular events in males and females
Approach 1: An NCBI BLAST of COL3A1 in humans and COL1A1 in Zebrafish will be conducted and the FASTA sequences retrieved and aligned by Clustal Omega software. A conserved single amino acid residue within the collagen triple helix repeat protein domain will be identified for mutation. Using Base Editing, the identified point-mutation will be generated in a Zebrafish model. Male and female wildtype Zebrafish, COL1A1 mutant Zebrafish with a known vEDS phenotype (COL1A1 c.934C>T) [3], and the newly identified mutation will be used as the study arms. The vasculature of each organism will be visualized and measured in each sex group for signs of aneurysms and vascular abnormalities. The Hypothesis is that mutations in the alpha triple-helix region will result in a dysfunctional COL1A1 protein leading to weakened vasculature. The Rationale is that the COL1A1 homolog in Zebrafish will be an effective model as it contains the same protein domains relating to fibril collagen synthesis in vEDS.
Aim 2: Examine sex-differences in differential gene expression for disease vascular phenotypes
Approach 2: RNA-seq will be conducted on aortic tissue for both wild type male and female zebrafish and those harboring the COL1A1 vEDS mutation. This will be done in juvenile Zebrafish developmentally as that is when presentation of vEDS sex differences are the highest. The gene expression profiles can be stratified by sex and sorted by integrin and collagen binding GO terms. Genes that are differentially expressed between sexes and wildtype/disease Zebrafish could indicate different molecular mechanisms in disease. These genes could then be induced in Zebrafish using a CRISPR/Cas9 system to study disease phenotype. The Hypothesis is that there will be differential gene expression in aortic vascular wall tissue relating to integrin and collagen binding between male and female zebrafish with vEDS. The Rationale is that males and females present with different disease phenotypes at an early age, and RNA-seq will allow for gene expression differences within aortic wall tissues to be analyzed.
Aim 3: Examine sex-differences in integrin and collagen binding proteins for vEDS
Approach 3: Integrins are critical for ECM cell-wall adhesion and attach to the subendothelial ECM when a vascular injury occurs [4]. Each of the male and female mutant zebrafish, along with wild type controls, will have aortic tissue harvested to examine the presence of integrins and collagen binding proteins. The aortic tissues would be chemically tagged in an iTRAQ system, and the proteins detected through mass spectrometry. For functional analysis, the proteins present will be sorted by GO terms regarding ECM and integrins, which are expected to be less enriched in males compared to females. Knockouts of relevant genes will be completed in male zebrafish to confirm functionality. The Hypothesis is that males will have a decreased presence of collagen binding proteins and integrins which help protect against injury during vasculature rupturing. The Rationale is that integrins bind to motifs on the collagen sequence in COL1A1, and vEDS mutations have been shown to hinder integrin binding ability and functionality.
References
[1] The Ehlers-Danlos Society. (2017). What are the Ehlers-Danlos Syndromes? | The Ehlers Danlos Society. The Ehlers Danlos Society. https://www.ehlers-danlos.com/what-is-eds/
[2] Byers PH. Vascular Ehlers-Danlos Syndrome. 1999 Sep 2 [Updated 2019 Feb 21]. In: Adam MP, Feldman J, Mirzaa GM, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2024. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1494/
[3] Cortini, F., Marinelli, B., Romi, S., Seresini, A., Pesatori, A.C., Seia, M., Montano, N., & Bassotti, A. (2017). A New COL3A1 Mutation in Ehlers-Danlos Syndrome Vascular Type With Different Phenotypes in the Same Family. Vascular and Endovascular Surgery, 51, 141 - 145.
[4] Hoop, Cody L. et al., Molecular underpinnings of integrin binding to collagen-mimetic peptides containing vascular Ehlers–Danlos syndrome–associated substitutions in Journal of Biological Chemistry, Volume 294, Issue 39, 14442 - 14453
Drafts of Specific aims
stack03172024SpecificAimsdraft1.doc |
stackkate04162024specificaimsdraft2_.docx |
stackkate05092024specificaimsfinal.doc |