BAKGRUND: I Sverige är cancer en av de vanligaste sjukdomarna. Bröstcancer är den vanligaste cancersjukdomen hos kvinnor. Bröstcancer kan sprida sig i kroppen och bilda en sekundär cancer som kallas metastas. Transformerande tillväxtfaktor-β-receptorer (TGF-β-receptorer) har en viktig roll i tumörutveckling. SYFTE: Syftet med denna studie är att studera rollen av TGF-β-receptorer i cancer, särskild bröstcancer med fokus på metastaserna. METOD: Den är en litteraturstudie, som är baserad på sex vetenskapliga artiklar. Dessa artiklar hämtas från databas PubMed genom användning av olika specifika sökord. RESULTAT: Resultat visar att TGF-β-receptorer har två motsatta rollar i cancerutveckling. Första fungerar TGF-β-receptorer som en tumörundertryckare i början av cancerutveckling. Däremot bidrar TGF-β-receptorer till utveckling av maligna celler lite senare under cancerutveckling. Det gör det genom att förbättra metastatiska potential samt undertrycka antitumörimmunitet. Metastaser bildas via Epithelial to mesenchymal transition med hjälp av vissa andra faktorer såsom cancerassocierade fibroblaster. Epithelial to mesenchymal transition och andel av cancerassocierade fibroblaster kan stimuleras av TGF-β-receptorer, som kan i sin tur leda till ökning av metastaseringen. Ett exempel på metastas är skelettcancer. DISKUSSION: Transformerande tillväxtfaktor-β receptorer, som kan påverka cancer, betraktas som ett bra fynd i läkemedelsutveckling mot cancer. TGF-β-receptorer har en viktig roll i tumörutveckling, särskilt sekundär cancer. Sekundär cancer/metastas är farligare än primär cancer, därför är det bättre att försöka förstå hela mekanismen bakom TGF-β-receptorer på ett tydligt sätt, för att kunna behandla patienter i god tid. SLUTSATS: Transformerande tillväxtfaktor- β -receptorer är en av de viktigaste parametrarna för att få den optimala effekten av medicineringen mot cancer.

The v-rel oncogene is the most efficiently transforming member of the Rel/NF-κB family of transcription factors. Identification of genes or signal transduction pathways that contribute to v-Rel transformation provide insight into the mechanisms of tumorigenesis by Rel/NF-κB proteins. In these studies, the contribution of TGF-β/Smad signaling to v-Rel transformation was assessed. TGF-β/Smad signaling regulates several cellular processes, including growth, differentiation, and apoptosis and has been implicated in a number of different cancers. Using microarray technology and Northern blot analysis, key components of the TGF-β/Smad pathway (tgf-β2 and tgf-β3 ligands, TGF-β type II receptor, and receptor-activated smad3) were identified with upregulated mRNA expression in v-Rel-transformed fibroblasts and lymphoid cells relative to control cells. A corresponding change in their protein levels was also observed. Further analysis revealed elevated levels of the phosphorylated, active form of Smad3, which correlated with its increased DNA-binding activity in v-Rel transformed cells. In contrast, the overexpression of c-Rel resulted in little to no alteration in the RNA and protein expression of members of the TGF-β/Smad pathway. Further studies demonstrated that elevated TGF-β/Smad signaling is required for the transforming ability of v-Rel. Blocking TGF-β signaling with a kinase inhibitor of TGF-β type I receptor inhibited the activation of Smad3 and dramatically reduced the ability of v-Rel transformed cells to form colonies in soft agar. Overexpression of a constitutively active form of Smad3 in the inhibitor-treated cells restored their ability to form colonies in soft agar close to the levels seen in untreated cells. Additional experiments with dominant negative Smad3 also revealed its ability to hinder the oncogenic potential of v-Rel. In complementary experiments, a stimulatory effect on v-Rel transformation was observed with cells treated with recombinant TGF-β2 ligand or overexpressed with wild-type Smad3. Taken together, these studies demonstrate that TGF-β signaling is crucial for the transformation potential of v-Rel and is primarily mediated by Smad3 activity. / text

Doctor of Philosophy (PhD) / Background: The development of the blood supply in the primate retina has been extensively studied; however the relationship of the differentiating retina to the choroidal blood supply is less well known. The interaction of astrocytes and vascular endothelial cells promotes the development of the retinal vasculature from 14 weeks’ gestation (WG). Initially, astrocytes lead the developing capillaries from the optic nerve towards the macular area. However, neither astrocytes nor endothelial cells enter a prescribed avascular area, within which the fovea later forms. This may be attributed to expression of a factor that inhibits astrocyte and endothelial cell proliferation in the fovea. A factor found in the CNS that is already known to have these effects is transforming growth factor-β (TGF-β). Aims: This thesis investigated the relationship between retinal maturation and choroidal blood vessel supply and the possible role for TGF-β as an antiangiogenic factor in maintaining an avascular fovea during primate retinal development. Methods: Human eyes between 11 WG and 40 years were obtained with ethical approval from Prince of Wales Hospital and the NSW Lions Eye Bank and fixed and sectioned for histological procedures or prepared for polymerase chain reaction (PCR). Macaque eyes from foetal day (fd) 64 to postnatal 11years (p11y) were obtained from Bogor Agriculture University, Indonesia with the approval of the Ethics Committee of the University of Washington, Seattle, USA. Macaque eyes were also fixed and sectioned for immunohistochemistry and in situ hybridisation. RNA was extracted from human foetal retinas and used for RTPCR (Reverse Transcriptase PCR), QPCR (Quantitative PCR) and preparation of riboprobes. PCR products were analysed using both restriction digest and sequencing. RTPCR was used to identify TGF-β1, TGF-β2 and TGF-β3 in the developing human and in the developing and adult macaque retinas whilst QPCR was used to quantify the TGF-β isoforms in central compared to peripheral retina and in foetal compared to adult retina. In situ hybridisation was performed according to a standard protocol and visualised using Roche HNPP Fast Red detection set with designed riboprobes for TGF-β1, TGF-β2 and TGF-β3 (DIG RNA labelling kit). Some sections were counterstained with vimentin antibody. Immunohistochemistry was performed on human retina and choroid sections using antibodies to CD34 and Ki67 and on human and macaque retina using antibodies to synaptophysin, vimentin, GFAP, calbindin, S-opsin, RG-opsin, rhodopsin, TGF-β1, TGF-β2, TGF-β3 and their receptors TβRI and TβRII. Sections of the retina were imaged and analysed using either a Leica Confocal microscope and TCSNT software or Zeiss Confocal microscope and LSM 5 Pascal software. Data from the human retina and choroid sections corresponding to different regions (foveal, parafoveal nasal, parafoveal temporal, nasal and temporal) was collected to measure the number of Ki-67 immunolabelled mitotic endothelial cells and the area of CD34 immunolabelled choriocapillaris using Adobe Photoshop version 5.0.2, NIH software version 1.62 (measurement macros) and Excel. In the human and macaque sections the intensity of TGF-β protein and mRNA expression was captured from different regions of the retina (foveal, parafoveal nasal, parafoveal temporal, nasal, temporal, nasal to disc) to compile montages. Montages were then re-imported into LSM 5 Pascal software to measure the optical density across each montage along the ganglion cell layer, outer neuroblastic zone and photoreceptor layer collecting data in Excel for graphical representation. In addition to the montages, individual sections were assessed for co-localisation of TGF-β and TβR to various retinal cell types. Results: Analyses of choriocapillaris area and endothelial cell (EC) proliferation were able to demonstrate that the area of choriocapillaris endothelium is greater in the foveal region at all ages (14-18.5WG), that the rate of choriocapillaris EC proliferation declines dramatically over this same period and that the lowest rates of EC proliferation are at the incipient fovea. Most importantly these findings indicate that EC proliferation in the choriocapillaris does not appear to be promoted by increased metabolic activity in central retinal neurons which are more developed with higher oxygen and nutrient demands, which is the mechanism widely thought to regulate development of the retinal vasculature. PCR showed all TGF-β isoforms to be present in the human developing and adult retina. QPCR revealed that TGF-β2 was the most predominant isoform, followed by TGF-β3 with very small amounts of TGF-β1 seen. The isoforms were more abundant in developing rather than adult retina and in central rather than peripheral retina. Studies of the distribution of TGF-β protein and mRNA using immunohistochemistry and in situ hybridisation confirmed the low levels of TGF-β1 protein and mRNA observed in QPCR and demonstrated distinct centroperipheral gradients in the photoreceptor layer for TGF-β2 and TGF-β3. Relative high amounts of TGF-β in the fovea could affect vascular patterning due to TβRI seen in astrocytes which lead the blood vessels at the foveal rim at the level of the ganglion cell plexus. TGF-β2 and TGF-β3 expression is detected before formation of the foveal avascular zone (FAZ) at fd64 (~15WG) - fd73 (~17WG) with levels peaking in the foveal region at fd105 (~25WG) by the time the FAZ forms. Conclusions: This thesis has shown that EC proliferation in the choriocapillaris does not appear to be promoted by increased metabolic activity in central retinal neurons as reduced rates of EC proliferation in the ‘foveal’ chorioretinal location were observed at all ages studied between 14 and 18.5WG. The findings suggest that mechanisms regulating proliferation and growth of the choroidal vasculature are independent of differentiation in the neural retina and are therefore different to those governing the formation of the retinal vasculature. All TGF-β isoforms are expressed in developing and adult human and macaque retina with TGF-β2 being the predominant isoform. TGF-β isoforms are more abundant in central compared to peripheral retina and in developing compared to adult retina. Centro-peripheral gradients of TGF-β2 and TGF-β3 across the photoreceptor layer and TβRI on astrocytes support the presence of TGF-β in the fovea as an antiproliferative and antiangiogenic factor by helping to define the FAZ early in development, well before 23-25 WG in humans and before fd100 in macaques.

TGF-β negatively affects the maintenance and expansion of hematopoietic stem cells ex vivo and its inhibition has been widely studied as a treatment for numerous hematopoietic disorders and cancers. Current inhibitory strategies (small molecule ATP competitors and neutralizing antibodies) are compared to a novel cell-permeable peptide-based inhibitor of TGF-β RI. Multiple levels of assay from biochemical to functional are utilized with the aim of applying the most successful inhibitor to hematopoietic stem cell culture. The neutralizing antibody proved ineffective in the short-term biochemical assay but was extremely effective at neutralizing TGF-β signalling in proliferation and hematopoietic colony-forming cell assays with no evidence of toxicity. The small molecule inhibitors (SD-208 and Pyrazole TGF-β RI inhibitor) were equally effective at micromolar levels in all forms of assay, with SD-208 being slightly more potent. The novel peptide inhibitor proved ineffective in all assays, which is likely a result of its rapid degradation in live cells.

TGF-β negatively affects the maintenance and expansion of hematopoietic stem cells ex vivo and its inhibition has been widely studied as a treatment for numerous hematopoietic disorders and cancers. Current inhibitory strategies (small molecule ATP competitors and neutralizing antibodies) are compared to a novel cell-permeable peptide-based inhibitor of TGF-β RI. Multiple levels of assay from biochemical to functional are utilized with the aim of applying the most successful inhibitor to hematopoietic stem cell culture. The neutralizing antibody proved ineffective in the short-term biochemical assay but was extremely effective at neutralizing TGF-β signalling in proliferation and hematopoietic colony-forming cell assays with no evidence of toxicity. The small molecule inhibitors (SD-208 and Pyrazole TGF-β RI inhibitor) were equally effective at micromolar levels in all forms of assay, with SD-208 being slightly more potent. The novel peptide inhibitor proved ineffective in all assays, which is likely a result of its rapid degradation in live cells.

Breast cancer is the most common cancer in female population worldwide. In addition to mutations, the breast tumor microenvironment especially the tumor cell - stroma interactions through extracellular matrix components and multiple growth factors have been shown to promote tumor progression. Among those, increases in both TGF-β (transforming growth factor beta) activities and periostin expression were associated with tumor cell survival, proliferation and metastasis. TGF-β role in breast cancer progression including its ability to promote periostin expression has been extensively studied. In contrast, the role of periostin in cancer progression remains to be fully understood. Thus, the present study aimed to determine whether TGF-β and periostin have effect on each other’s expressions in breast tumor and stroma cells using in vitro cell models. Through Western blot analyses and ELISAs, the periostin and TGF-β expressions of both stroma and tumor cells were analyzed following TGF-β and periostin treatments, respectively. The results indicate that TGF-β treatments led to significant increase in periostin expression in fibroblasts (p<0.05). In addition, periostin was differentially expressed by human breast cancer cells following TGF-β1 treatment. The TGF-β activities involved activation of pSMAD2 in both L929 fibroblasts and MCF10A mammary cells. Taken together, all experimental data indicate that within the breast tumor TGF-β and periostin likely participate in a regulation loop. Whether this putative regulation loop is critical to metastasis remains to be determined. Should periostin play a critical role in breast cancer progression, it could become a specific target in the preventive and/or therapeutic development of breast cancer patients.

Doctor of Philosophy (PhD) / Background: The development of the blood supply in the primate retina has been extensively studied; however the relationship of the differentiating retina to the choroidal blood supply is less well known. The interaction of astrocytes and vascular endothelial cells promotes the development of the retinal vasculature from 14 weeks’ gestation (WG). Initially, astrocytes lead the developing capillaries from the optic nerve towards the macular area. However, neither astrocytes nor endothelial cells enter a prescribed avascular area, within which the fovea later forms. This may be attributed to expression of a factor that inhibits astrocyte and endothelial cell proliferation in the fovea. A factor found in the CNS that is already known to have these effects is transforming growth factor-β (TGF-β). Aims: This thesis investigated the relationship between retinal maturation and choroidal blood vessel supply and the possible role for TGF-β as an antiangiogenic factor in maintaining an avascular fovea during primate retinal development. Methods: Human eyes between 11 WG and 40 years were obtained with ethical approval from Prince of Wales Hospital and the NSW Lions Eye Bank and fixed and sectioned for histological procedures or prepared for polymerase chain reaction (PCR). Macaque eyes from foetal day (fd) 64 to postnatal 11years (p11y) were obtained from Bogor Agriculture University, Indonesia with the approval of the Ethics Committee of the University of Washington, Seattle, USA. Macaque eyes were also fixed and sectioned for immunohistochemistry and in situ hybridisation. RNA was extracted from human foetal retinas and used for RTPCR (Reverse Transcriptase PCR), QPCR (Quantitative PCR) and preparation of riboprobes. PCR products were analysed using both restriction digest and sequencing. RTPCR was used to identify TGF-β1, TGF-β2 and TGF-β3 in the developing human and in the developing and adult macaque retinas whilst QPCR was used to quantify the TGF-β isoforms in central compared to peripheral retina and in foetal compared to adult retina. In situ hybridisation was performed according to a standard protocol and visualised using Roche HNPP Fast Red detection set with designed riboprobes for TGF-β1, TGF-β2 and TGF-β3 (DIG RNA labelling kit). Some sections were counterstained with vimentin antibody. Immunohistochemistry was performed on human retina and choroid sections using antibodies to CD34 and Ki67 and on human and macaque retina using antibodies to synaptophysin, vimentin, GFAP, calbindin, S-opsin, RG-opsin, rhodopsin, TGF-β1, TGF-β2, TGF-β3 and their receptors TβRI and TβRII. Sections of the retina were imaged and analysed using either a Leica Confocal microscope and TCSNT software or Zeiss Confocal microscope and LSM 5 Pascal software. Data from the human retina and choroid sections corresponding to different regions (foveal, parafoveal nasal, parafoveal temporal, nasal and temporal) was collected to measure the number of Ki-67 immunolabelled mitotic endothelial cells and the area of CD34 immunolabelled choriocapillaris using Adobe Photoshop version 5.0.2, NIH software version 1.62 (measurement macros) and Excel. In the human and macaque sections the intensity of TGF-β protein and mRNA expression was captured from different regions of the retina (foveal, parafoveal nasal, parafoveal temporal, nasal, temporal, nasal to disc) to compile montages. Montages were then re-imported into LSM 5 Pascal software to measure the optical density across each montage along the ganglion cell layer, outer neuroblastic zone and photoreceptor layer collecting data in Excel for graphical representation. In addition to the montages, individual sections were assessed for co-localisation of TGF-β and TβR to various retinal cell types. Results: Analyses of choriocapillaris area and endothelial cell (EC) proliferation were able to demonstrate that the area of choriocapillaris endothelium is greater in the foveal region at all ages (14-18.5WG), that the rate of choriocapillaris EC proliferation declines dramatically over this same period and that the lowest rates of EC proliferation are at the incipient fovea. Most importantly these findings indicate that EC proliferation in the choriocapillaris does not appear to be promoted by increased metabolic activity in central retinal neurons which are more developed with higher oxygen and nutrient demands, which is the mechanism widely thought to regulate development of the retinal vasculature. PCR showed all TGF-β isoforms to be present in the human developing and adult retina. QPCR revealed that TGF-β2 was the most predominant isoform, followed by TGF-β3 with very small amounts of TGF-β1 seen. The isoforms were more abundant in developing rather than adult retina and in central rather than peripheral retina. Studies of the distribution of TGF-β protein and mRNA using immunohistochemistry and in situ hybridisation confirmed the low levels of TGF-β1 protein and mRNA observed in QPCR and demonstrated distinct centroperipheral gradients in the photoreceptor layer for TGF-β2 and TGF-β3. Relative high amounts of TGF-β in the fovea could affect vascular patterning due to TβRI seen in astrocytes which lead the blood vessels at the foveal rim at the level of the ganglion cell plexus. TGF-β2 and TGF-β3 expression is detected before formation of the foveal avascular zone (FAZ) at fd64 (~15WG) - fd73 (~17WG) with levels peaking in the foveal region at fd105 (~25WG) by the time the FAZ forms. Conclusions: This thesis has shown that EC proliferation in the choriocapillaris does not appear to be promoted by increased metabolic activity in central retinal neurons as reduced rates of EC proliferation in the ‘foveal’ chorioretinal location were observed at all ages studied between 14 and 18.5WG. The findings suggest that mechanisms regulating proliferation and growth of the choroidal vasculature are independent of differentiation in the neural retina and are therefore different to those governing the formation of the retinal vasculature. All TGF-β isoforms are expressed in developing and adult human and macaque retina with TGF-β2 being the predominant isoform. TGF-β isoforms are more abundant in central compared to peripheral retina and in developing compared to adult retina. Centro-peripheral gradients of TGF-β2 and TGF-β3 across the photoreceptor layer and TβRI on astrocytes support the presence of TGF-β in the fovea as an antiproliferative and antiangiogenic factor by helping to define the FAZ early in development, well before 23-25 WG in humans and before fd100 in macaques.

Abstract Dentin forms the hard tissue portion of the dentin-pulp complex, while the dental pulp is soft connective tissue that retains the vitality of the dentin. Odontoblasts form the outermost cell layer of pulp and play a central role during dentin formation by producing and mineralizing the dentin matrix. The understanding of the defensive reactions in the dentin-pulp complex is limited. Information about the transcriptome and proteome of pulp tissue and odontoblasts would facilitate understanding of their functions during health and disease. The aim of this study was to investigate the expression profiles of human pulp tissue and odontoblasts in vivo and in vitro using large-scale expression analysis methods. Also, the suitability of these methods in pulp biological research in vivo and in vitro was evaluated. cDNA microarray revealed only minor variation and 2-D electrophoresis combined with mass spectrometry revealed no differences between healthy and carious teeth pulp tissue in vivo. The effect of transforming growth factor β1 (TGF-β1) on pulp and odontoblasts was studied in vitro using oligonucleotide-based microarrays, and marked changes in the transcriptome were revealed, especially in the expression of chemokine- and cytokine-related genes. Transiently increased interleukin expression was confirmed at the protein level by antibody array. DNA microarray analysis of native pulp tissue and odontoblasts was used to search for potential odontoblast markers. Only one gene related to extracellular matrix organization and biogenesis, matrilin 4, and two expressed sequence tags (ESTs), which represent transcribed sequences encoding possibly unknown genes, were identified in odontoblasts but not in pulp. Analysis of mature native odontoblasts and cultured odontoblast-like cells by DNA microarray revealed a high similarity (84%) between native and cultured cells. Also, differential expression levels of selected neuronal proteins were observed and confirmed at the mRNA and protein levels. In conclusion, microarray is a powerful tool for pulp biology, especially for in vitro studies. TGF-β1 was revealed as a potent regulator of proinflammatory responses in the dentin–pulp complex. In addition, several potential odontoblast markers were identified by microarray, and the similarity of cultured odontoblast-like cells used in the study with native odontoblasts was confirmed.

La voie de signalisation TGF-β joue un rôle biphasique durant la cancérogenèse. Mon laboratoire a identifié une nouvelle protéine inhibitrice de la voie TGF-β, WWP1. WWP1 est une E3 ubiquitine ligase qui induit la polyubiquitination et la dégradation du récepteur de type I au TGF-β. De plus, le gène WWP1 est amplifié dans une large proportion de cancers mammaires et prostatiques, suggérant que WWP1 pourrait jouer un rôle clé dans les processus de cancérogenèse liés au TGF-β. Mon projet de thèse était donc de caractériser la régulation de l’activité catalytique de WWP1 ainsi que son mécanisme d’action dans la cellule. Mes résultats montrent qu’à l’état basal, WWP1 est monoubiquitinée, son activité de polyubiquitination étant réduite par l’effet inhibiteur qu’exercent les domaines C2 et/ou WW sur son domaine HECT. En présence de substrats, la protéine WWP1 « s’ouvre » et peut alors induire la polyubiquitination et la dégradation de ses substrats. De plus, nous avons observé qu’un mutant de WWP1, détecté dans un cancer de la prostate, est incapable de s’autoréguler selon ce modèle. Il présente une plus forte activité ligase envers lui-même et ses substrats, ce qui entraîne une atténuation de la réponse cytostatique du TGF-β pouvant conférer une activité oncogénique à WWP1. De plus, nous avons identifié STARD13 comme un nouveau partenaire de WWP1. STARD13 est une protéine à activité RhoGAP, considérée comme un suppresseur de tumeur. Nous avons montré que STARD13 permet l’association de WWP1 avec la GTPase RhoA, entraînant ainsi la polyubiquitination et la dégradation de RhoA. De façon intéressante, le complexe WWP1/STARD13 est impliqué dans le remodelage de l’architecture du cytosquelette en dégradant préférentiellement la forme activée de RhoA. Ces résultats ont permis d’identifier un nouveau rôle de WWP1 qui pourrait jouer un rôle essentiel durant la migration des cellules cancéreuses lors du processus métastatique. La caractérisation de nouveaux mécanismes de régulation et d’action de WWP1 devrait permettre à terme d’identifier si WWP1 est un marqueur diagnostique dans le cancer et/ou une nouvelle cible thérapeutique pour le développement de médicaments anticancéreux. / The TGF-β pathway plays a biphasic role during cancerogenesis. My laboratory identified a new protein, WWP1, as a negative regulator of TGF-β signaling. WWP1 is an E3 ubiquitin ligase that triggers polyubiquitination and degradation of TGF-β type I receptor. A genomic amplification of WWP1 is found in a large portion of mammary and prostatic tumors, suggesting a key role for WWP1 during carcinogenesis related to TGF-β. My thesis project was to determine the regulation of the catalytic activity of WWP1 and a new molecular mechanism of action of WWP1 whose deregulation can be implicated in cancerogenesis. My results indicate that at steady states, WWP1 is monoubiquitinated, its polyubiquitination activity being silenced due to the inhibitory effects of C2 or/and WW domains on its Hect domain. In presence of substrates, WWP1 is « opened » and induces polyubiquitination and degradation of its substrates. Moreover, a WWP1 mutation found in prostate cancer disrupts this regulatory mechanism. It possesses an increased ligase activity towards itself and its substrates, which leads to the attenuation of TGF-β cytostatic signaling, a consequence that could conceivably confer tumorigenic properties to WWP1. We also identified STARD13 as a novel WWP1 interacting partner. STARD13 has a RhoGAP activity, and is considered as a tumor suppressor. We have shown that STARD13 mediates the association of WWP1 with the GTPase RhoA, ultimately leading to RhoA polyubiquitination and degradation. Interestingly, the WWP1/STARD13 complex is involved in the actin cytoskeleton rearrangement by preferentially targeting the active form of RhoA for degradation. These results reveal a previously unrecognized role for WWP1, which could play a key role in the migration of cancer cells during metastasis. Characterization of new regulation and action mechanisms for WWP1 should allow identifying whether WWP1 is a diagnosis biomarker in cancer and/or a new therapeutic target for the development of anticancer drugs.

The prevalence of cardiovascular disease in the United States dramatically increases with age. A hallmark feature of the aged myocardium is increased fibrosis resulting in diastolic dysfunction. Moreover, the survival of patients subsequent to a myocardial infarction is inversely related to age because of a certain extent to maladaptive remodeling mediated by cardiac fibroblasts. Our hypothesis is that cardiac fibroblast (CF) dysfunction results in overexpressed TGF-β1 leading to increased cardiac collagen content in the aged population. TGF-β1 stimulates the synthesis of the extracellular matrix proteins, including collagen in the cardiac tissues. The RT-PCR analysis of mRNA expression of TGF-β1 of the CF was increased by 43% in the aged mice as compared to the younger. The stiffness of the left ventricle is expressed with the slope of the end-diastolic pressure-volume relationship parameter, β (mmHg/μL). In a mouse model, we demonstrated that β was 0.30 ± 0.05 in the young as compared to 0.52 ± 0.10 in the aged (p < .05). The ventricular stiffness was associated with the myocardial collagen content; namely, young versus the aged was 9.5 ± 4.0 as compared to 16.4 ± 2.3% of total protein, respectively (p < .05). In conclusion, the gene structure-function relationships support our hypothesis that cardiac fibroblast disregulation contributes to diastolic filling dysfunction in elderly persons. These data provide a potential contributory mechanism for diastolic dysfunction that may be vital in caring for the aged open-heart surgical patient.