Role of fibroblast growth factor receptors (FGFR) and FGFR like-1 (FGFRL1) in mesenchymal stromal cell differentiation to osteoblasts and adipocytes.

Fibroblast growth factors (FGF) and their receptors (FGFRs) regulate many developmental processes including differentiation of mesenchymal stromal cells (MSC). We developed two MSC lines capable of differentiating to osteoblasts and adipocytes and studied the role of FGFRs in this process. We identified FGFR2 and fibroblast growth factor receptor like-1 (FGFRL1) as possible actors in MSC differentiation with gene microarray and qRT-PCR. FGFR2 and FGFRL1 mRNA expression strongly increased during MSC differentiation to osteoblasts. FGF2 treatment, resulting in downregulation of FGFR2, or silencing FGFR2 expression with siRNAs inhibited osteoblast differentiation. During adipocyte differentiation expression of FGFR1 and FGFRL1 increased and was down-regulated by FGF2. FGFR1 knockdown inhibited adipocyte differentiation. Silencing FGFR2 and FGFR1 in MSCs was associated with decreased FGFRL1 expression in osteoblasts and adipocytes, respectively. Our results suggest that FGFR1 and FGFR2 regulate FGFRL1 expression. FGFRL1 may mediate or modulate FGFR regulation of MSC differentiation together with FGFR2 in osteoblastic and FGFR1 in adipocytic lineage.

Osteocyte-derived HB-GAM (pleiotrophin) is associated with bone formation and mechanical loading.

HB-GAM (also known as pleiotrophin) is a cell matrix-associated protein that is highly expressed in bone. It affects osteoblast function, and might therefore play a role in bone development and remodeling. We aimed to investigate the role of HB-GAM in bone in vivo and in vitro. The bones of HB-GAM deficient mice with an inbred mouse background were studied by histological, histomorphometrical, radiological, biomechanical and mu-CT analyses and the effect of immobilization was evaluated. HB-GAM localization in vivo was studied. MLO-Y4 osteocytes were subjected to fluid shear stress in vitro, and gene and protein expression were studied by subtractive hybridization, quantitative PCR and Western blot. Human osteoclasts were cultured in the presence of rhHB-GAM and their formation and resorption activities were assayed. In agreement with previous reports, the skeletal structure of the HB-GAM knockout mice developed normally. However, a growth retardation of the weight-bearing bones was observed by 2 months of age, suggesting a link to physical activity. Adult HB-GAM deficient mice were characterized by low bone formation and osteopenia, as well as resistance to immobilization-dependent bone remodeling. HB-GAM was localized around osteocytes and their processes in vivo and furthermore, osteocytic HB-GAM expression was upregulated by mechanical loading in vitro. HB-GAM did not affect on human osteoclast formation or resorption in vitro. Taken together, our results suggest that HB-GAM is an osteocyte-derived factor that could participate in mediating the osteogenic effects of mechanical loading on bone.

Selective augmentation of histone H1 phosphorylation sites by interaction of poly(ADP-ribose) polymerase and cdc2-kinase: comparison with protein kinase C.

The molecular interactions between PARP I, cdc2-kinase, PKC and histone H1 were determined with the aid of the common phosphate acceptor function of histone H1 to both kinases. PKC phosphorylates both histone H1 and PARP I and PARP I augments the acceptor function of histone H1. When both acceptors (PARP I and histone H1) are present an apparent distributive phosphorylation of both acceptors takes place. In contrast, cdc2-kinase only phosphorylates histone H1, and the activation of this reaction by PARP I does not involve PARP I-cdc2-kinase binding only PARP I-histone H1 association. Since the phosphorylation of histone H1 by PKC is a model reaction with no apparent physiologic consequences, the PARP I activated phosphorylation of histone H1 by cdc2-kinase, by contrast, reflects a physiologically meaningful regulation of the linker histone by a cyclin dependent kinase (cdc2-kinase). The increased phosphorylation of histone H1 by cdc2-kinase following PARP I-histone H1 binding results in the appearance of new phosphorylated histone H1 polypeptides as measured by proteolytic digestion and re-electrophoresis of cdc2-kinase phosphorylated polypeptides, indicating a probable conformational change in histone H1, following PARP I binding. The cell biologic significance of this reaction in PARP I ligand-induced enzyme induction is briefly analysed.

Molecular interactions between poly(ADP-ribose) polymerase (PARP I) and topoisomerase I (Topo I): identification of topology of binding.

The molecular interactions of poly(ADP-ribose) polymerase I (PARP I) and topoisomerase I (Topo I) have been determined by the analysis of physical binding of the two proteins and some of their polypeptide components and by the effect of PARP I on the enzymatic catalysis of Topo I. Direct association of Topo I and PARP I as well as the binding of two Topo I polypeptides to PARP I are demonstrated. The effect of PARP I on the 'global' Topo I reaction (scission and religation), and the activation of Topo I by the 36 kDa polypeptide of PARP I and catalytic modifications by poly(ADP-ribosyl)ation are also shown. The covalent binding of Topo I to circular DNA is activated by PARP I similar to the degree of activation of the 'global' Topo I reaction, whereas the religation of DNA is unaffected by PARP I. The geometry of PARP I-Topo I interaction compared to automodified PARP I was reconstructed from direct binding assays between glutathione S-transferase fusion polypeptides of Topo I and PARP I demonstrating highly selective binding, which was correlated with amino acid sequences and with the 'C clamp' model derived from X-ray crystallography.

Activation of topoisomerase I by poly [ADP-ribose] polymerase.

Poly(ADP-ribose) polymerase (PARP I) and Topoisomerase I (Topo I) were reisolated from calf thymus to eliminate cross contamination as tested by immunotransblots. The specific activity of Topo I was greatly increased by added PARP I, following saturation kinetics. Recombinant PARP I and isolated PARP I at final purity were indistinguishable in terms of their activation of Topo I. There was a coincidence of experimentally obtained binding constants and computer generated values based on the kinetic model, indicating that the association of PARP I and Topo I is rate limiting in the catalytic activation of Topo I by PARP I. Polypeptide domains of PARP I that are required for protein-protein binding and protein-DNA binding also activate Topo I. Fluorescence resonance energy transfer between fluorophor-labeled PARP I and Topo I was demonstrated. The binding of Topo I to circular SV40 DNA, assayed either by the formation of a) the sum of non-covalently and covalently attached Topo I to DNA or b) by the covalently bound transient intermediate in the presence of camptothecin, was augmented when PARP I protein was bound to SV40 DNA. These binding experiments provide a molecular basis for the kinetic activation of Topo I by PARP I inasmuch as the increased superhelicity of SV40 DNA induced by PARP I may facilitate the formation of a more Topo I-DNA complex that increases the rate of the DNA breakage-reunion cycle of Topo I catalysis.

Unusual potentiation by vinca alkaloids of the cytostatic and cytocidal action of methyl-3,5-diiodo-4-(4'-methoxyphenoxy) benzoate (DIME) and its nonhydrolyzable ethanone analog (DIPE) on MDA-MB-231 human mammary cancer cells.

Drug interaction between DIME or DIPE ¿1-[3, 5-diiodo-4-(4'-methoxyphenoxy)-phenyl]-ethanone¿ with vincristine and vinblastine on the growth rate of MDA-MB-231 human mammary cancer cells was determined by the median effect kinetic method. Mutually exclusive cellular binding sites were identified kinetically and isobologram analyses showed potentiation. The combind effect of 0.75 MICROM DIME and 2 nM vincristine demonstrated a nearly type of mutual activation. It was shown that the nonhydrolyzable DIME derivative DIPE is equivalent to DIME, but because of its biological stability is a preferred drug candidate. Vinblastine-DIME cooperative action is similar to that of vincristine-DIME (or DIPE). Activation of caspase 3 by both DIME and vincristine is greatly potentiated when both drugs are added simultaneously in a given proportion. We propose that following a primary binding of DIME and vinca alkaloids to microtubules, an as yet unrecognized mutual activation of caspase 3 apoptotic path is initiated, explaining DNA fragmentation and cell death. A subpopulation of cancer cells, capable of slow growth at 1.5 microM DIME was identified. This cell type was also killed by the DIME-vincristine drug combination.

Isolation and identification of a proteinase from calf thymus that cleaves poly(ADP-ribose) polymerase and histone H1.

A proteinase was isolated from calf thymus that degraded pADPRT, histone H1 and alpha-casein in a Ca(2+)-dependent manner. In a five-step procedure, a homogenous proteinase was obtained with a subunit structure of 80 and 30 kDa. The amino-acid homology of an internal sequence as well as kinetic and inhibitor assays identified the proteinase as calpain I. It is suggested that even though the general substrate alpha-casein is widely used for the assaying of calpains, more appropriately physiological cellular components (pADPRT and histone H1) specify the thymus proteinase.

Inhibition of migration of MDA-MB-231 cells by methyl-3,5-diiodo-4-(4'-methoxyphenoxy)benzoate (DIME).

The GTPase activity of purified dimeric tubulin (alpha+beta) at 5 mu M was insensitive to methyl-3,5-diiodo-4-(4'-methoxyphenoxy) benzoate (DIME), in contrast to nocodazole which activated GTPase. Cellular motility of MDA-MB-231 (human mammary cancer) cells migrating through 12-mu m pores was inhibited by DIME similar to nocodazole in a drug concentration-and DIME structure-dependent manner. An increase of cytoplasmic ATPase activity of DIME-treated cells without a decrease in ATP contents of intact cells suggests that DIME may also influence additional as yet unidentified ATP-dependent system(s) probably also involved in cell motility. These results show that DIME not only arrests cells in M phase but also inhibits cell motility in interphase. However the cellular mode of action of DIME is different from the action of other toxic tubulin-targeted drugs, despite the fact that DIME in a concentration-dependent manner disrupts microtubule structures in intact cells.

Structural specificity and tumoricidal action of methyl-3,5-diiodo-4-(4'-methoxyphenoxy) benzoate (DIME).

Seventeen homologs and analogs of methyl-3,5-diiodo-4(4'-methoxyphenoxy) benzoate (DIME), a hormonally inactive analog of thyroid hormones, have been synthesized and their antitumor activity scored by assaying their antitumorigenic effect in vivo following pretreatment of E-ras 20 cells with the drug. In vivo feeding of DIME in large doses had a similar antitumor effect on human tumor xenogafts in vivo without noticeable toxicity of DIME. Inhibition of clonogenicity with MDA-MB-231 cells by DIME yielded I-50 values similar to those found in tests measuring cell growth inhibition (median I-50 less than 1.0 mu M) The apparent ultimate signal for cytocidal action of DIME is the development of dose-dependent double strand cuts of DNA. All human tumor cells so far tested, with the exception of A-549 cells (lung cancer), show high sensitivity to DIME (I-50 1.0 mu M or below). The partially refractory behavior of A-549 cells to DIME is due to the presence of an esterase that cleaves the methyl ester group of DIME. Other tumor cells have negligible esterase activity, while such activity in homogenates of normal mouse tissues is high. Cells in culture take up DIME and the magnitude of uptake parallels drug sensitivity to DIME. Extrapolation from the correlations between drug efficacy, drug uptake, esterase activity and the absence of significant in vivo toxicity of DIME point to uptake of DIME by cells in culture but not by normal cells operating in intact organs in vivo. In contrast, tumor cells in vivo take up DIME and succumb to its cytocidal action just like cells in culture.

Inhibition of the GTP-dependent polymerization of tubulin by methyl-3,5-diiodo-4-(4'-methoxyphenoxy) benzoate (DIME).

The hormonally inactive thyroid hormone analog methyl-3,5-diiodo-4-(4'-methoxyphenoxy) benzoate (DIME) at 1-5 mu M concentrations inhibits the GTP-dependent polymerization of MTP as determined by an optical test. This inhibition is critically dependent on the concentration of GTP. The quantitative correlation between the concentrations of DIME and GTP, under conditions of a linear rat of MTP polymerization, follows Michaelis-Menten kinetics and the inhibition portrays a 'mixed' type, where k(m) for GTP and V-max are altered simultaneously. Chemical analogs of DIME inhibit MTP polymerization parallel to their antitumorigenic action in vivo. The MTP site is one of the early cellular response sites of DIME.

Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-1,2-benzopyrone (INH2BP).

Bovine aortic endothelial cells were converted to a highly tumorigenic cell line by transfection with Ha-ras and stimulation with thrombin. Sustained pretreatment with a non-cytotoxic concentration (600 mu M) of 5-iodo-6-amino-1,2-benzopyrone (INH2BP), a lipophilic ligand of poly(ADP-ribose) polymerase, abrogated in vivo tumorigenicity, of 10(5) cells per inoculum an effect which developed progressively during 2 to 6 weeks of drug treatment. The initial action of the drug was cytostasis, consisting of an arrest in prophase, extreme cell enlargement consistent with cytoplasmic hypertrophy, as seen by EM, and dramatic morphologic changes. Although neither DNA, RNA or protein syntheses are directly affected by INH2BP, apparently newly synthesized cellular DNA is degraded by endonucleases, which are upregulated by the inhibition of their poly-ADP-ribosylation. The drug treated cells exhibited greatly increased respiration and aerobic glycolysis, due to an augmentation of,glycolytic and respiratory enzymes in enlarged cells. These responses to the drug were reversible in cell cultures following drug removal, within 5-10 days drug exposure but the progressive loss of tumorigenicity in nude mice that developed after 3-6 weeks of drug exposure of cells, prior to inoculation to nude mice, was not reversible in vivo. Drug treatment produced a sustained 70-80% inhibition of pADPRT in intact cells at 600 mu M extracellular concentration of INH2BP. The prerequisite for the abrogation of tumorigenicity was the maintenance of pADPRT inhibition. The arrest of cell multiplication and a large decrease of Topo I, especially of Topo II and MAP kinase activities occurred without loss of enzyme protein as assayed in cell extracts of drug-treated cells. However INH2BP had no direct effect on these enzymes. Drug treatment down-regulated DNA-methyltransferase, PKC, ODC proteins, diminished cyclin A protein, but the hypophosphorylated form of Rb protein was significantly augmented. None of the enzymatic components of signal pathways so far studied, were directly affected by INH2BP. The inhibition of pADPRT by INH2BP coincided with an induction or activation of alkaline phosphatase and leucyl and glutamyl peptidase. The pADPRT content or the expression of pADPRT gene were not influenced by drug treatment, but the expression of ras gene was completely absent in nontumorigenic drug-treated cells, without a loss of ras gene from genomic DNA. Telomerase activity was not directly influenced by INH2BP treatment when assayed in diluted cell extracts, but the addition of homogeneous pADPRT to cell extracts, to approach physiological concentration of this protein in the cell, inhibited telomerase activity by binding of the polymer-free pADPRT to telomer templates. We conclude that inhibition of pADPRT indirectly down-regulates growth stimulatory signal pathways and sustains growth-arrested cells in culture at a pre-apoptotic threshold which explains the absence of tumorigenicity in vivo.

Potential chemotherapeutic activity of 4-iodo-3-nitrobenzamide. Metabolic reduction to the 3-nitroso derivative and induction of cell death in tumor cells in culture.

A C-nitroso prodrug, 4-iodo-3-nitrobenzamide, was synthesized, and its action on a variety of tumor cells of human and animal origin tested. This prodrug was reduced transiently by tumor cells to 4-iodo-3-nitrosobenzamide at a very low rate, which was, however, sufficient to kill tumor cells. The final reduction product was 4-iodo-3-aminobenzamide, and no intermediates accumulated. No toxicity could be observed in hamsters even at 200 mg/kg, given i.p. daily for 7 days. The chemical reactivity of both 4-iodo-3-nitrosobenzamide and its noniodinated homolog with reduced ascorbate yielded the hydroxylamines. With glutathione, 4-iodo-3-aminobenzamide was formed, suggesting glutathione sulfinic acid formation. Confirming earlier studies, 4-iodo-3-nitrosobenzamide inactivated poly(ADP-ribose) polymerase by zinc ejection from the first zinc finger of this nuclear protein. The iodinated nitroso compound was more effective than its iodine-free analog. Selective tumoricidal action appeared to correlate with the reduction of the nitro group to nitroso in tumor cells, and with the previously described subsequent induction of tumor apoptosis by the C-nitroso intermediate. These processes were accelerated by buthionine sulfoximine, which diminishes cellular GSH.

Identification of domains of poly(ADP-ribose) polymerase for protein binding and self-association.

Cellular proteins extracted from normal and cancer cells bind polymerizing ADP-ribose transferase (pADPRT) on nitrocellulose membrane transblots. Histones at 1 mg/ml concentration completely prevent the binding of pADPRT to cellular proteins, indicating that the binding of histones to pADPRT sites competitively blocks the association of pADPRT to proteins other than histones. The direct binding of pADPRT to histones is shown by cross-linking with glutaraldehyde. The COOH-terminal basic histone H1 tail binds to the basic polypeptide domain of pADPRT. The basic domain present in the NH2-terminal part of core histones is the probable common structural feature of all core histones that accounts for their binding to pADPRT. Two polypeptide domains of pADPRT were identified, by way of CNBr fragments, to bind histones. These two domains are located within the 64-kDa fragment of pADPRT and are contiguous with the polypeptide domains that were shown to participate in self-association of pADPRT, ending at the 606th amino acid residue. The polypeptide domains of pADPRT which participate in DNA binding are thus shown to associate also with other proteins. Intact pADPRT binds to both the zinc-free or zinc-reconstituted basic polypeptide fragments of pADPRT. Histones activate auto-poly(ADP)-ribosylation of pADPRT by increasing the number of short oligomers on pADPRT. This reaction is also dependent in a biphasic manner on the concentration of pADPRT. Histones in solution are only marginally poly(ADP)-ribosylated but are good polymer acceptors when incorporated into artificial nucleosome structures.

Induction of endonuclease-mediated apoptosis in tumor cells by C-nitroso-substituted ligands of poly(ADP-ribose) polymerase.

6-Nitroso-1,2-benzopyrone and 3-nitrosobenzamide, two C-nitroso compounds that inactivate the eukaryotic nuclear protein poly(ADP-ribose) polymerase [NAD+:poly(adenosine diphosphate D-ribose) ADP-D-ribosyltransferase, ADPRT, EC 2.4.2.30] at one zinc-finger site, completely suppressed the proliferation of leukemic and other malignant human cells and subsequently produced cell death. Tumoricidal concentrations of the drugs were relatively harmless to normal bone marrow progenitor cells and to superoxide formation by neutrophil granulocytes. The cellular mechanism elicited by the C-nitroso compounds consists of apoptosis due to DNA degradation by the nuclear calcium/magnesium-dependent endonuclease. This endonuclease is maintained in a latent form by poly(ADP-ribosyl)ation, but inactivation of ADPRT by C-nitroso drugs derepresses the DNA-degrading activity. ADPRT is thus identified as a critical regulatory enzyme component of a DNA-binding multiprotein system that plays a central function in defining DNA structures in the intact cell.

Inhibition of HIV-1 IIIb replication in AA-2 and MT-2 cells in culture by two ligands of poly (ADP-ribose) polymerase: 6-amino-1,2-benzopyrone and 5-iodo-6-amino-1,2-benzopyrone.

The effects of two adenosine diphosphoribose transferase (ADPRT) enzyme inhibitory ligands, 6-amino-1,2-benzopyrone and its 5-iodo-derivative, were determined in AA-2 and MT-2 cell cultures on the replication of HIV-1 IIIb, assayed by an immunochemical test for the HIV protein p24, and syncytium formation, characteristic of HIV-infected cells. Intracellular concentrations of both drugs were sufficient to inhibit poly(ADP-ribose) polymerase activity within the intact cell. Both drugs inhibited HIV replication parallel to their inhibitory potency on ADPRT, but distinct differences were ascertained between the two cell lines. In AA-2 cells both p24 and syncytium formation were depressed simultaneously, whereas in MT-2 cells only syncytium formation was inhibited by the drugs, and the p24 production, which remained unchanged during viral growth, was unaffected. Both drugs only moderately depressed the growth rate of the AA-2 and MT-2 cells and there was no detectable cellular toxicity. Results suggest the feasibility of the development of a new line of ADPRT ligand anti-HIV drugs that fundamentally differ in their mode of action from currently used chemotherapeutics.

Inhibitory binding of adenosine diphosphoribosyl transferase to the DNA primer site of reverse transcriptase templates.

Purified adenosine diphosphoribose transferase protein binds to RNA-DNA hybrid templates of reverse transcriptase at the DNA primer site and inhibits RT activity of HIV and MMu RTs. This action is prevented by auto-poly-ADP-ribosylation of the transferase but is reinduced by inhibitory ligands of the enzyme.

Destabilization of Zn2+ coordination in ADP-ribose transferase (polymerizing) by 6-nitroso-1,2-benzopyrone coincidental with inactivation of the polymerase but not the DNA binding function.

6-Nitroso-1,2-benzopyrone, an oxidation product of 6-amino-1,2-benzopyrone, binds to the DNA-recognizing domain of the ADP-ribose transferase protein and preferentially destabilizes Zn2+ from one of the two zinc finger polypeptide complexes present in the intact enzyme, as determined by the loss of 50% of 65Zn2+ from the 65Zn(2+)-isolated protein molecule, coincidental with the loss of 99% of enzymatic activity. The 50% zinc-deficient enzyme still binds to a DNA template, consisting of a 17-mer DNA primer annealed to M13 positive strand, resulting in the blocking of DNA synthesis by the Klenow fragment of Pol I. Auto-poly-ADP-ribosylated ADP-ribose transferase, which is the probable physiological state of this protein in intact cells, does not bind to primer-template DNA and does not block DNA synthesis by the Klenow fragment. On the basis of this in vitro model it is proposed that molecules which inhibit or inactivate ADP-ribose transferase in intact cells can induce significant alteration in DNA structure and replication.

Evidence for the participation of histidine residues located in the 56 kDa C-terminal polypeptide domain of ADP-ribosyl transferase in its catalytic activity.

Purified ADPRT protein was inactivated by the histidine specific reagent diethylpyrocarbonate, binding to two histidine residues, or by a relatively histidine selective photoinactivation method. Inactivation with up to 1.3 mM diethylpyrocarbonate was reversible by hydroxylamine. Enzymatic inactivation coincided with the loss of binding capacity of the enzyme protein to benzamide affinity matrix but not to DNA cellulose. Labelled diethylpyrocarbonate was identified exclusively in the 56 kDa carboxyl-terminal polypeptide where 2 out of 13 histidine residues were modified by this reagent. It is proposed that histidine residues in the 56 kDa polypeptide may participate as initiator sites for polyADP-ribosylation.

Macromolecular association of ADP-ribosyltransferase and its correlation with enzymic activity.

The macromolecular self-association of ADP-ribosyltransferase protein in solution was studied by several experimental techniques: quantitative gel filtration, electrophoretic analyses in non-denaturing gels, and cross-linking the enzyme protein with glutaraldehyde, dimethyl pimelimidate, dimethyl suberimidate, dimethyl 3,3'-dithiobisproprionimidate and tetranitromethane. The self-association of the polypeptide components obtained by plasmin digestion was also determined by using the above cross-linking agents. Monomers and cross-linked dimers of the enzyme protein, possessing enzymic activity, were separated in non-denaturing gels by electrophoresis. The basic polypeptide fragments, exhibiting molecular masses of 29 kDa and 36 kDa, self-associated, whereas the polypeptides with molecular masses of 56 kDa and 42 kDa associated only to a negligible extent, indicating that the peptide regions that also bind DNA and histones are probable sites of self-association in the intact enzyme molecule. Macromolecular association of the enzyme was indicated by a protein-concentration-dependent red-shift in protein fluorescence. The specific enzymic activity of the isolated ADP-ribosyltransferase depended on the concentration of the enzyme protein, and at 2.00 microM concentration the enzyme was self-inhibitory. Dilution of the enzyme protein to 30-40 nM resulted in a large increase in its specific activity. Further dilution to 1-3 nM coincided with a marked decrease of specific activity. Direct enzymic assays of electrophoretically separated monomers and cross-linked dimers demonstrated that the dimer appears to be the active molecular species that catalyses poly(ADP-ribose) synthesis. The NAD+ glycohydrolase activity of the enzyme was also dependent on protein concentration and was highest at 1-3 nM enzyme concentration, when polymerase activity was minimal, indicating that the monomeric enzyme behaved as a glycohydrolase, whereas poly(ADP-ribosyl)ation of enzyme molecules was maximal when the enzyme tends to be self-associated to the dimeric form.

Influence of the measles virus on the proliferation and protein synthesis of aortic endothelial and smooth muscle cells.

To clarify whether some viruses could influence the different functions and membrane permeability of the aortic cells, we have examined in a model experiment the in vitro effect of the measles virus on the aortic endothelial and smooth muscle cells. The aortic cells infected with the virus failed to reveal gross cytopathic effect. Occasionally, however, syncytium formation and nuclear inclusions were observed. In infected endothelial cells lysosome containing viral nucleocapsids were seen. The early phase of measles virus replication inhibited the proliferation of endothelial cells of all species tested, while uniformly stimulated the replication of the smooth muscle cells relative to the control. In bovine aortic endothelial and smooth muscle cells the protein synthesis had been suppressed by the 4th to 6th hours postinfection. The results indicate that measles virus infection may be among the risk factors of atherosclerosis. It may damage endothelial cells by altering the cell membrane permeability and could induce proliferation of aortic smooth muscle cells.