Abstract Introduction The role of cardiac lymphatic system in myocardial infarction (MI) is still unclear. A new method to detect and characterize MI without contrast agent is a relaxation along a fictitious field in nth rotating frame (RAFFn). The RAFFn takes advantage of the fictitious magnetic field, which is produced by a fast sweep of an effective radio frequency field, to increase a spin locking field strength without increasing the specific absorption rate. MI is detected as increased RAFFn relaxation times and cardiac edema by an increased T2 relaxation time. We have shown earlier that MI size can be accurately measured by the RAFFn relaxation times. Purpose To study the effects of the lack of cardiac lymphatic system on MI and cardiac edema in a mouse model. Methods Transgenic (TG) mice expressing soluble decoy VEGF receptor 3 (sVEGFR3) thus blocking lymphatic vessel formation in the heart and wild type (WT) control mice were used. MI was induced in 13–17 week old TG (n=11) and WT (n=14) mice by ligating the left anterior descending coronary artery. The RAFFn (TRAFF2 and TRAFF4), a continuous wave T1ρ and a T2 relaxation times were acquired at time points 0, 3, 7 and 21 days after the MI at 9.4 T. Histological sections were stained with hematoxylin eosin and Sirius red to assess cellularity and MI area. An Area of difference (AOD) was determined by subtracting MI areas based on TRAFF2, TRAFF4 and T1ρ maps from MI area based on T2 maps. Results MI size based on the TRAFF4 and T2 relaxation time maps were larger at early time points 3 and 7 days post MI in the WT group compared to the TG group (Figure 1A-B, p<0.05). However, the MI size was significantly larger in the T2 relaxation time map in the TG group compared to the WT group at the last time point and interaction between the groups were significant as a function of time (Figure 1A-B, p<0.05). The AOD values, which reflect cardiac edema, increased in the TG group as a function of time (Figure 1C, p<0.001). TRAFF2, TRAFF4 (Figure 1D), T1ρ and T2 relaxation times increased significantly (≈50%, p<0.001) after the MI compared to remote areas in both groups. In the WT group, the lymphatic vessel network is fully functional and removes edema efficiently between days 3 and 21 after the MI, while in the TG group the MI area in T2 map is relative stable indicating insufficient edema removal, caused by the lymphatic deficiency and insufficient lymphangiogenesis in the TG group. The MIs were also verified based on Sirius red stained histology (Figure 1E). Figure 1 Conclusion Lymphatic deficiency increases cardiac edema (AOD values) 7–21 days after MI as compared to the WT group. Results support the importance of cardiac lymphatic vessels for healing after MI. Effects of the lymphatics on MI can be detected based on the MI size difference based on the TRAFFn and the T2 relaxation times. Acknowledgement/Funding Doctoral Programme of Molecular Medicine

Malignant gliomas (MGs) are the most common malignant primary brain tumors with a short life estimate accompanied by a marked reduction in the quality of life. Herpes Simplex virus-1 thymidine kinase ganciclovir (HSV-TK/GCV) system is the best characterized enzyme prodrug therapy in use. However, lipophobicity of GCV and low enzymatic activity of HSV-TK reduce the treatment efficacy. Tomato TK (ToTK) has shown high activity in combination with its specific substrate azidothymidine (AZT). The aim of this study was to evaluate whether ToTK/AZT could be used as an alternative to HSV-TK/GCV therapy. Both treatments demonstrated cytotoxicity in human MG cells in vitro. In vivo, both treatments decreased tumor growth and tumors were smaller in comparison with controls in mouse orthotopic MG model. Survival of ToTK/AZT-treated mice was significantly increased compared with control mice (*P<0.05) but not as compared with HSV-TK/GCV-treated mice. No significant differences were observed in clinical chemistry safety analyses. We conclude that both treatments showed a beneficial treatment response in comparison to controls on tumor growth and ToTK/AZT also on survival. There were no significant differences between these treatments. Therefore ToTK/AZT could be considered as an alternative treatment option for MG because of its favorable therapeutic characteristics.

Purpose: Lymphatic vessels take part in lipid absorption, inflammatory reactions and draining of large blood vessels. Even though these processes are involved in arterial diseases, the role of lymphatic vessels in atherosclerosis has not been clarified yet. In this study, we evaluated the effect of insufficient lymphatic function on the lipoprotein metabolism and development of atherosclerosis by blocking Vascular Endothelial Growth Factor Receptor 3 (VEGFR3), the key mediator of lymphangiogenesis. Methods: Mice expressing soluble VEGFR3 (sVEGFR3) were cross-bred with atherosclerotic LDLR-/-/ApoB100/100 mice. Mice were fed with chow diet for 3-4 months, 7-8 months and 11-12 months followed by high-fat diet (42% of calories from fat and 0.15% from cholesterol) up to 12 weeks. Lipid levels and lipoprotein profiles were analyzed from blood plasma. Atherosclerotic lesion size was determined from en face aortas and cross-sections of aortic arches and lesion composition was evaluated with modified Movat's staining and immunohistochemical stainings for macrophages (mMQ), blood vessels (CD31) and lymphatic vessels (podoplanin). Results: Compared to LDLR-/-/ApoB100/100 controls, sVEGFR3 x LDLR-/-/ApoB100/100 mice had significantly increased plasma cholesterol levels with chow diet (8.2±0.4 mmol/l vs. 16.3±1.2 mmol/l, respectively) and up to 6 weeks with high-fat diet (36.9±3.0 mmol/l vs. 50.8±4.4 mmol/l, respectively). In addition, sVEGFR3 x LDLR-/-/ApoB100/100 mice had higher VLDL and LDL cholesterol and triglyceride levels than controls. Especially in young sVEGFR3 x LDLR-/-/ApoB100/100 mice, elevated cholesterol levels induced accelerated atherogenesis characterized by increased lesion size and rapid accumulation of foam cells and cholesterol crystals into the lesions. Even though the number of vasa vasorum in the atherosclerotic lesions was at similar level in the study and control groups, lymphatic vessels were almost absent in the lesions of sVEGFR3 x LDLR-/-/ApoB100/100 mice. Conclusions: Here we show that lipoprotein metabolism and atherogenesis are affected by insufficient lymphatic draining. These results suggest that lymphatic vessels have a previously undiscovered role in the lipid transport and highlight a potential target for the treatment of lipid related diseases, such as atherosclerosis.

Lentiviruses have shown great promise for human gene therapy. However, no optimal strategies are yet available for noninvasive imaging of virus biodistribution and subsequent transduction in vivo. We have developed a dual-imaging strategy based on avidin–biotin system allowing easy exchange of the surface ligand on HIV-derived lentivirus envelope. This was achieved by displaying avidin or streptavidin fused to the transmembrane anchor of vesicular stomatitis virus G protein on gp64-pseudotyped envelopes. Avidin and streptavidin were efficiently incorporated on virus particles, which consequently showed binding to biotin in ELISA. These vectors, conjugated to biotinylated radionuclides and engineered to express a ferritin transgene, enabled for the first-time dual imaging of virus biodistribution and transduction pattern by single-photon emission computed tomography and magnetic resonance imaging after stereotactic injection into rat brain. In addition, vector retargeting to cancer cells overexpressing CD46, epidermal growth factor and transferrin receptors using biotinylated ligands and antibodies was demonstrated in vitro. In conclusion, we have generated novel lentivirus vectors for noninvasive imaging and targeting of lentivirus-mediated gene delivery. This study suggests that these novel vectors could be applicable for the treatment of central nervous system disorders and cancer.

The effects of glucocorticoids and retinoids on connective tissue biosynthesis were studied in cultured human skin fibroblasts (HSFs). More specifically attention was paid to the effects of dexamethasone and 13-cis-retinoic acid (RA) on total protein and collagen synthesis and on collagen and fibronectin mRNA levels. The results indicated that dexamethasone reduced the relative collagen synthesis and collagen mRNA levels in HSFs and increased the total incorporation of proline into proteins, the latter effect being due to increased activity in the intracellular proline pool. 13-cis-RA did not affect collagen synthesis at the concentration studied (10(-7) M) but it did reduce the corresponding mRNA levels. Simultaneous addition of both dexamethasone and 13-cis-RA or etretinate resulted in the largest decrease in type I and type III procollagen mRNA levels, indicating that retinoids do not oppose the effect of glucocorticoids on collagen synthesis in cultured HSFs. For comparison the effects of dexamethasone and 13-cis-RA on the mRNA levels of another extracellular matrix component, fibronectin, and of a constitutive enzyme, glyceraldehyde-3-phosphate dehydrogenase, were also studied. The results indicated, that dexamethasone treatment did not alter fibronectin mRNA levels in HSFs, while 13-cis-RA did so to a marked extent. Both dexamethasone and 13-cis-RA also reduced the mRNA level of glyceraldehyde-3-phosphate dehydrogenase, indicating that glucocorticoids and retinoids have both similar and different effects on gene expression in HSF.

Fibrosis is a hallmark symptom in a number of human diseases, including scleroderma, lung fibrosis, liver cirrhosis, atherosclerosis, osteoarthritis, and keloids. The most prominent biochemical manifestation of fibrotic lesions is an abnormal accumulation of extracellular matrix components, including type I and III collagen in mesenchymal tissues. This abnormal accumulation often results in severe malfunction of the affected tissues. It is important to note that the formation of fibrous tissue can also be a normal physiological response as it occurs, for instance, in wound healing. This suggests that in fibrotic diseases regulation of this normal physiological process is altered. A study of the mechanisms regulating the normal response should help in understanding the abnormal control of this process.

Glucocorticoids modulate various cellular functions such as proliferation, energy metabolism and the synthesis of proteins. In the present study, the response of collagen genes to dexamethasone in different stages of chick embryo development was studied in tendon and heart using Northern blot analysis and specific cDNA probes. The changes in collagen gene expression were compared to alterations in two reference mRNAs: actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The levels of specific mRNAs measured per ribosomal RNA in tendon and heart varied markedly during normal development. In tendon the relative levels of alpha-1(I), alpha-2(I) and alpha-1(III) collagen mRNAs were highest between days 14-16 when also the synthesis of matrix proteins is most active. In heart the levels of these mRNAs peaked at day 12. In addition, qualitative differences were observed in the expression of actin genes between tendon and heart. Dexamethasone in high dose decreased collagen mRNA levels in tendons, while in heart a stimulatory effect was noted. Dexamethasone also decreased GAPDH mRNA levels in tendons. The alterations in gene expression after dexamethasone treatment in tendon and heart did not correlate with the level of specific glucocorticoid receptors, which varied markedly during the development of chick embryos. The cDNA for pro alpha-1(I) collagen hybridized to two transcripts corresponding to 6.2 and 5.1 kb in tendon and heart. During normal development of chick embryos the ratio of 6.2/5.1 kb mRNAs decreased markedly in heart, but no such change was observed in tendons. Dexamethasone, however, decreased the ratio of 6.2/5.1 kb transcripts in tendons. There was a significant correlation between the ratio 6.2/5.1 kb transcripts and total alpha-1(I) mRNA both in tendon and heart, suggesting that the 6.2 kb transcript may be associated with the rate of synthesis of type I collagen.

A characteristic feature of fibroblasts cultured from affected skin areas of patients with systemic sclerosis (SSc) and localized scleroderma (morphea) is excessive activation of collagen biosynthesis. To elucidate the nature of fibroblast activation in scleroderma we have studied the expression of 3 noncollagenous connective tissue components, osteonectin, small dermatan sulfate proteoglycan (proteoglycan II, decorin), and transforming growth factor-beta 1 (TGF-beta 1), by measuring their mRNA levels in fibroblast cultures from 6 patients with SSc and 3 with morphea. A clear correlation was observed between the increase in type I collagen and osteonectin mRNA in these cell lines. The apparent overproduction of osteonectin by scleroderma fibroblasts is in accordance with the suggested activation of osteonectin expression during tissue remodeling. The levels of decorin mRNA showed marked variation in the cell lines, but were in no correlation with collagen or osteonectin mRNA. The levels of TGF-beta 1 mRNA were found to be slightly elevated in fibroblasts grown from affected scleroderma skin. This may suggest that this potent activator of collagen production has a role during the initial activation of dermal fibroblasts both in SSc and morphea.

Five human skin fibroblast lines were studied for type I collagen production and type I procollagen mRNA levels through the different growth phases. The cells were plated at low density and followed for 11 days at daily intervals through the stages of rapid growth and visual confluency until the cultures reached stationary growth phase. Each day one culture flask was labeled with [3H]proline for 24 h, and analyzed for production of radiolabeled type I collagen into culture medium. The cell layers were counted and subjected to isolation of cytoplasmic RNA and determination of type I procollagen mRNA levels. The results revealed an approx. 2-fold increase in procollagen production and mRNA levels when the cells reached visual confluency. Thereafter the synthesis rates and mRNA levels remained relatively constant, although a decreasing tendency of both parameters was observed upon further culturing. The results confirm that determination of cell density is important when cell cultures are used for measurement of collagen synthesis or mRNA levels. For determination of proα2(I) collagen mRNA an 1193 bp cDNA clone was constructed using RNA extracted from human fetal calvaria. Sequencing of the clone revealed some nucleotide and amino acid differences between the previously published sequences. This suggests the presence of more individual variation in procollagen coding sequences than expected.

CCAAT-binding factor (CBF) is a heteromeric mammalian transcription factor which binds to sequences containing a CCAAT motif in a number of promoters such as those for type I collagen, albumin, MHC Class II, beta-actin, and others. It consists of two different components that are both needed for DNA binding. We have purified the "A" chain of CBF to apparent homogeneity by sequence-specific DNA affinity chromatography followed by Mono S and Mono Q ion-exchange chromatography and obtained the amino acid sequences of tryptic peptides of this polypeptide. Amino acid sequences of two of these tryptic peptides were used to synthesize oligonucleotide primers. The primers served to obtain a small cDNA by the polymerase chain reaction method, which was then further used to obtain larger cDNA clones. DNA sequence analysis of a representative cDNA clone revealed the presence of an open reading frame of 207 amino acids coding for a putative polypeptide of 25 kDa. Transcription of these cDNAs in vitro followed by translation in a reticulocyte lysate produced a polypeptide that migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the same mobility as the native A chain. The deduced amino acid sequence of the A chain showed a remarkable identity over a length of 90-amino acid residues with a sequence of the Hap3 polypeptide, a component of a heteromeric multisubunit yeast transcription factor.

Increased gene transcription is a mechanism responsible for the exaggerated accumulation of type I and III collagens in scleroderma and other fibrotic syndromes. We review recent studies on the mechanisms that control transcription of the alpha 1 (I) and alpha 2 (I) collagen genes. Several specific DNA binding proteins have been identified which either activate or inhibit transcription of these genes. It is probable that the complexity of transcription factors that control the type I collagen genes corresponds to a multiplicity of cytokines and other hormonal effectors, which influence the expression of these genes by binding to specific receptors and by activating intracellular signaling pathways.

Annals of the New York Academy of SciencesVolume 580, Issue 1 p. 88-96 Transcriptional Mechanisms Controlling Types I and III Collagen Genes BENOIT DE CROMBRUGGHE, BENOIT DE CROMBRUGGHE Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030Search for more papers by this authorGERARD KARSENTY, GERARD KARSENTY Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030Search for more papers by this authorSANKAR MAITY, SANKAR MAITY Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030Search for more papers by this authorTUULA VUORIO, TUULA VUORIO Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030Search for more papers by this authorPELLEGRINO ROSSI, PELLEGRINO ROSSI Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030Search for more papers by this authorE. CRISTY RUTESHOUSER, E. CRISTY RUTESHOUSER Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030Search for more papers by this authorSANDRA H. McKINNEY, SANDRA H. McKINNEY Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030Search for more papers by this authorGUILLERMINA LOZANO, GUILLERMINA LOZANO Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030Search for more papers by this author BENOIT DE CROMBRUGGHE, BENOIT DE CROMBRUGGHE Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030Search for more papers by this authorGERARD KARSENTY, GERARD KARSENTY Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030Search for more papers by this authorSANKAR MAITY, SANKAR MAITY Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030Search for more papers by this authorTUULA VUORIO, TUULA VUORIO Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030Search for more papers by this authorPELLEGRINO ROSSI, PELLEGRINO ROSSI Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030Search for more papers by this authorE. CRISTY RUTESHOUSER, E. CRISTY RUTESHOUSER Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030Search for more papers by this authorSANDRA H. McKINNEY, SANDRA H. McKINNEY Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030Search for more papers by this authorGUILLERMINA LOZANO, GUILLERMINA LOZANO Department of Molecular Genetics The University of Texas M. D. Anderson Cancer Center Houston, Texas 77030Search for more papers by this author First published: February 1990 https://doi.org/10.1111/j.1749-6632.1990.tb17921.xCitations: 13AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume580, Issue1Structure, Molecular Biology, and Pathology of CollagenFebruary 1990Pages 88-96 RelatedInformation

CBF is a heteromeric mammalian transcription factor that binds to CCAAT sequences in a number of promoters such as the two type I collagen promoters, the albumin promoter, the major histocompatibility complex class II promoter, and others. It is composed of two components, A and B, that are both needed for DNA binding. We have isolated a rat cDNA containing the complete 341-amino acid coding sequence of the B component of CBF. Expression of this cDNA in vitro generates a polypeptide that shows the same dependency on the A component as the native B component in the formation of a complex with a CCAAT-containing DNA. The C-terminal portion of the B component from residue 260 to residue 312 shows a 75% sequence identity with a portion of the Hap2 protein, a component of a heteromeric CCAAT-binding protein in yeast. In contrast, the rest of the protein shows little sequence homology with Hap2, although both proteins contain glutamine-rich domains. In the B component of CBF this domain spans the amino-terminal 60% of the protein, whereas in Hap2 this domain is much smaller. Hence, only a few changes in one domain of this protein were tolerated during evolution between yeast and mammals, whereas the rest of the protein diverged much more extensively.

The effects of 13-cis retinoic acid (RA) and dexamethasone on the levels of epidermal growth factor (EGF) receptor and fibroblast derived proteoglycan core protein (PG40) mRNAs were studied in human skin fibroblasts. The EGF receptor is involved in the regulation of cellular proliferation and the synthesis of matrix proteins, and proteoglycan 40 is important for cell attachment and interaction with collagen and fibronectin. 13-cis-RA at a concentration of 10(-7) M markedly reduced the levels of the EGF receptor and PG40 mRNAs, the decreases being 33 and 56%, respectively. Dexamethasone reduced these mRNAs markedly less. Simultaneous treatment of the fibroblasts with 13-cis-RA and dexamethasone resulted in similar decreases in EGF receptor and PG40 mRNAs as with 13-cis-RA alone. Surprisingly, the proliferation rate of the fibroblasts was increased in the presence of dexamethasone under conditions similar to those which caused slight decrease in the EGF receptor mRNA levels. This indicates that glucocorticoids also affect the cellular growth by mechanisms which do not involve EGF receptors.

A cDNA clone for human proα(III) collagen mRNA was isolated from a cDNA library constructed for human fetal skin RNA. The clone, pHFS3, was identified by restriction mapping and sequencing. Comparison with previously published human type III collagen sequences revealed some differences which may reflect individual variation. The clone was used to study the expression of type III collagen mRNA in various fetal tissues in comparison to the expression of type I collagen mRNAs. In 15-18-week fetal skin the ratio of α (I) to α (III) collagen mRNAs was 0.8. Diaphyseal and calvarial bone contained high amounts of type I collagen mRNA and low levels of type III collagen mRNA, resulting in high type I/type III ratios. In situ hybridization of sections of skeletal tissues was employed to identif y the cells containing the mRNAs for types I, II and III procollagens. The results revealed differential expression patterns for these three collagen types in various human fetal tissues. Lack of coordinate expression suggests that production of type I and type III collagens is under different regulatory mechanisms in developing skeletal tissues.

The activation of collagen synthesis during development of silicotic fibrosis was studied in rats exposed, in dusting chambers, to respirable SiO2 for periods of 2, 4, 6 or 12 months. Control animals were exposed similarly to clean air or TiO2. Development of fibrosis was followed by histological examination, measurement of lung weight and determination of lung collagen content (as hydroxyproline). A steady increase in lung weight and collagen content together with changes in cellularity and metabolic activity of the lungs, as ascertained by chemical determination of DNA and RNA, were measured in the lungs of the SiO2-exposed animals. Hybridization of total lung RNA, extracted at each time point, with cDNA probes specific for type I and type III procollagen mRNA levels showed that the development of fibrosis was associated with increased levels, as compared to age matched controls, of pulmonary procollagen mRNAs. Interestingly, the highest levels of procollagen mRNAs were observed in young (pretreatment control) animals, suggesting that during pulmonary development collagen metabolism in lungs is even greater than during development of fibrosis. In rats exposed to SiO2 the increase in type III procollagen mRNA occurred earlier than the increase in type I procollagen mRNAs. These observations demonstrate both age-dependent and silicosis-related changes in pulmonary procollagen mRNA levels. The results suggest that development of silicosis is associated with an altered capacity of the lungs to regulate collagen accumulation.