David J. Rickard

Human Aortic Valve Calcification Is Associated With an Osteoblast Phenotype

Background—Calcific aortic stenosis is the third most common cardiovascular disease in the United States. We hypothesized that the mechanism for aortic valve calcification is similar to skeletal bone formation and that this process is mediated by an osteoblast-like phenotype. Methods and Results—To test this hypothesis, we examined calcified human aortic valves replaced at surgery (n=22) and normal human valves (n=20) removed at time of cardiac transplantation. Contact microradiography and micro-computerized tomography were used to assess the 2-dimensional and 3-dimensional extent of mineralization. Mineralization borders were identified with von Kossa and Goldner’s stains. Electron microscopy and energy-dispersive spectroscopy were performed for identification of bone ultrastructure and CaPO4 composition. To analyze for the osteoblast and bone markers, reverse transcriptase–polymerase chain reaction was performed on calcified versus normal human valves for osteopontin, bone sialoprotein, osteocalcin, alkaline phosphatase, and the osteoblast-specific transcription factor Cbfa1. Microradiography and micro-computerized tomography confirmed the presence of calcification in the valve. Special stains for hydroxyapatite and CaPO4 were positive in calcification margins. Electron microscopy identified mineralization, whereas energy-dispersive spectroscopy confirmed the presence of elemental CaPO4. Reverse transcriptase–polymerase chain reaction revealed increased mRNA levels of osteopontin, bone sialoprotein, osteocalcin, and Cbfa1 in the calcified valves. There was no change in alkaline phosphatase mRNA level but an increase in the protein expression in the diseased valves. Conclusions—These findings support the concept that aortic valve calcification is not a random degenerative process but an active regulated process associated with an osteoblast-like phenotype.

Estrogens and Progestins

Publisher Summary This chapter focuses on the structure and function of estrogens and progestins. The physiological actions of sex steroids contribute to sexual dimorphism of the skeleton, timing of epiphyseal closure, determination of peak bone mass, maintenance of mineral homeostasis during reproduction, and maintenance of bone mass, architecture, and mineral homeostasis in adults. Estrogen (E) is the major sex steroid that affects the growth, remodeling, and homeostasis of the skeleton. E regulates the processes of osteoblast (OB)-mediated bone formation and osteoclast (OC)-mediated bone resorption at multiple levels, which includes progenitor cell recruitment, proliferation, differentiation, and programmed cell death. Additionally, a second estrogen receptor distinct from the classical receptor has been identified, and loss-of-function mutations for these two receptor isoforms produce different skeletal phenotypes in mice. The activity of steroid nuclear receptors is modulated by the family of steroid receptor coregulators, which is composed of coactivators and corepressors. Coactivators, when bound to active receptor conformations, mediate favorable interactions with the basal transcriptional machinery, stabilize the preinitiation complex and, overall, stimulate gene transcription. Conversely, corepressors bind preferentially to inactive receptor conformations and prevent the interaction of the receptor with coactivators, thus resulting in nonproductive transcription factor complexes, which suppress gene transcription. Progesterone (P) is often given in conjunction with E during hormone replacement therapy of postmenopausal women to minimize some of the undesirable effects of E on reproductive tissues. P has been shown to stimulate mineralization of newly induced bone in rats and to increase the cortical bone formation rate in spayed Beagle dams.

Development and Characterization of Conditionally Immortalized Osteoblast Precursor Cell Lines from Human Bone Marrow Stroma

Although the differentiation of mature osteoblasts has been well studied, there is still a need for a convenient way to study preosteoblast differentiation. Our laboratory has recently described a method for isolating small numbers of authentic osteoblast precursor cells from human bone marrow (Rickard et al., J Bone Miner Res 11:312–324, 1996). Here we describe the conditional immortalization of these cells by retroviral transfection with the amphotrophic vector, pZipSV40tsa58, which encodes for a temperature‐sensitive mutant form of the simian virus large T‐antigen. At the permissive temperature of 34°C, the cell lines proliferated, but differentiation was arrested, whereas at the restrictive temperature of 39.5°C, proliferation was decreased and differentiation was induced. As assessed by semiquantitative reverse transcriptase PCR after 4 days of culture at 39.5°C, the six cell lines expressed similar mRNA levels both constitutively and in response to dexamethasone (Dex) and 1α,25‐dihydroxyvitamin D3 (1,25(OH2)D3) for osteoblast (alkaline phosphatase [ALP], type I collagen [Col I], osteocalcin [OC], and parathyroid hormone receptor [PTH‐R] and adipocyte (lipoprotein lipase [LPL]) genes. In the presence of 10−8 M Dex, gene expression for ALP, PTH‐R, and LPL increased, but that for OC decreased. Stimulation with 10−8 M 1,25(OH2)D3 increased gene expression for ALP, OC, and Col I. Changes in protein production for ALP, OC, and type I procollagen in response to Dex and 1,25(OH2)D3 were similar to changes in mRNA levels. When cultured at 39.5°C with ascorbate and β‐glycerolphosphate for 21 days, mineralization of matrix occurred, whereas culture with Dex plus 1,25(OH2)D3, or rabbit serum led to enhanced formation of cytoplasmic lipid droplets within 6 days. Thus, these cell lines are capable of bipotential differentiation and should serve as an excellent tool to study the molecular mechanisms that regulate and select for osteoblast and adipocyte differentiation in humans.

Phytoestrogen genistein acts as an estrogen agonist on human osteoblastic cells through estrogen receptors α and β

Genistein, a soybean isoflavone, has estrogen‐like activity in mammals, including the prevention of bone loss. However, whether its mechanism of action on bone turnover is distinct from that of estrogen or raloxifene is unknown. Although genistein has been reported to bind both estrogen receptor (ER) isoforms (α and β), little is known concerning differential activation of gene expression via these ER isoforms. To examine this question, comparison of the responses of normal fetal osteoblast (hFOB) cells stably expressing either ERα (hFOB/ERα9) or ERβ (hFOB/ERβ6), to treatment with genistein, 17β‐estradiol (E2) or raloxifene were conducted. In hFOB/ERα9 cells, both genistein and E2 increased the endogenous gene expression of the progesterone receptor (PR), the proteoglycan versican, and alkaline phosphatase (AP), but inhibited osteopontin (OP) gene expression and interleukin‐6 (IL‐6) protein levels. Raloxifene had no effect on these bone markers. Genistein, but not raloxifene, also mimicked E2 action in the hFOB/ERβ6 cells increasing PR gene expression and inhibiting IL‐6 production. To determine whether the gene regulatory actions of genistein in human osteoblast cells occur at the level of transcription, its action on the transcriptional activity of a PR‐A promoter‐reporter construct was assessed. Both genistein and E2 were found to stimulate the PR promoter in the hFOB cell line when transiently co‐transfected with either ERα or ERβ. Whereas hFOB cell proliferation was unaffected by E2, raloxifene or genistein at low concentrations, higher concentrations of genistein, displayed significant inhibition. Together, these findings demonstrate that genistein behaves as a weak E2 agonist in osteoblasts and can utilize both ERα and ERβ.

Bone morphogenetic protein-6 production in human osteoblastic cell lines. Selective regulation by estrogen.

Bone morphogenetic proteins (BMPs) induce differentiation of osteoblast and chondroblast lineage cells from uncommitted mesenchymal precursors. Because estrogen has potent osteochondrogenic actions, we investigated its effect on BMP production in two estrogen-responsive, human immortalized cell lines (hFOB/ER3 and hFOB/ER9) that display the mature osteoblast phenotype. These cell lines were produced by stable transfection of the estrogen receptor (ER) gene into immortalized fetal osteoblasts at low ( approximately 800 ER/ nucleus) and at high ( approximately 3, 900 ER/nucleus) levels, respectively. As assessed by reverse transcriptase PCR, treatment with 17beta-estradiol (10(-)10 - 10(-)7 M) increased steady-state levels of BMP-6 mRNA dose dependently by twofold in the hFOB/ER3 cells and by over threefold in the hFOB/ER9 cells. Messenger RNA levels for transforming growth factors-beta1 and -beta2 and BMPs-1 through -5 and -7 levels were unchanged. The results were confirmed by sequence determination of the PCR product and by Northern blot analysis for total RNA. 17beta-estradiol increased BMP-6 protein production sixfold by Western analysis. Cotreatment with antiestrogens (ICI 182,780 or 4-hydroxytamoxifen) antagonized the effects of 17beta-estradiol. These data suggest that some of the skeletal effects of estrogen on bone and cartilage may be mediated by increased production of BMP-6 by osteoblasts.

Molecular and cellular mechanisms of estrogen action on the skeleton.

The many recent and exciting advances that have taken place in the field of estrogen action on the skeleton are the subjects of this review. Leading these new developments is the discovery of alternative estrogen receptors that exhibit differential mechanisms of transcriptional control of estrogen‐responsive promoters, thereby broadening both the ranges of possible target cells and their responses. More potentially important genes under estrogenic control have been identified in vitro, and the skeletal phenotypes caused by disruption of estrogen signaling due to mutations in humans and mice have been described. Lastly, clinical studies in humans have revealed a greater appreciation for the importance of estrogen in bone mass maintenance in both sexes. J. Cell. Biochem. Suppls. 32/33:123–132, 1999.

Further characterization of human fetal osteoblastic hFOB 1.19 and hFOB/ERα cells: Bone formation in vivo and karyotype analysis using multicolor fluorescent in situ hybridization

We have previously generated an immortalized human fetal osteoblastic cell line (hFOB) using stably transfected temperature sensitive SV40 T‐antigen (Harris et al. [ 1995a ] J. Bone. Miner. Res. 10:178–1860). To characterize these cells for phenotypic/genotypic attributes desired for a good cell model system, we performed karyotype analysis by multicolor fluorescent in situ hybridization (M‐FISH), their ability to form bone in vivo without developing cell transformation, and finally their ability to form extracellular matrix formation in vitro. The karyotype analysis of hFOB cells revealed structural or numeric anomalies involving 1–2 chromosomes. In contrast, the human osteosarcoma MG63 cells displayed multiple, and often complex, numeric, and structural abnormalities. Subcutaneous injection of hFOB cells in the presence of Matrigel into nude mice resulted in bone formation after 2–3 weeks. Electron microscopic analysis of the extracellular matrix deposited by hFOB cells in culture revealed a parallel array of lightly banded fibrils typical of the fibrillar collagens such as type I and III. These results demonstrate that the hFOB cell line has minimal chromosome abnormalities, exhibit the matrix synthetic properties of differentiated osteoblasts, and are immortalized but non‐transformed cell line. These hFOB cells thus appear to be an excellent model system for the study of osteoblast biology in vitro. J. Cell. Biochem. 87: 9–15, 2002.

Estrogen regulation of human osteoblast function is determined by the stage of differentiation and the estrogen receptor isoform

Although osteoblasts have been shown to respond to estrogens and express both isoforms of the estrogen receptor (ERα and ERβ), the role each isoform plays in osteoblast cell function and differentiation is unknown. The two ER isoforms are known to differentially regulate estrogen‐inducible promoter‐reporter gene constructs, but their individual effects on endogenous gene expression in osteoblasts have not been reported. We compared the effects of 17β‐estradiol (E) and tamoxifen (TAM) on gene expression and matrix formation during the differentiation of human osteoblast cell lines stably expressing either ERα (hFOB/ERα9) or ERβ (hFOB/ERβ6). Expression of the appropriate ER isoform in these cells was confirmed by northern and western blotting and the responses to E in the hFOB/ERβ6 line were abolished by an ERβ‐specific inhibitor. The data demonstrate that (1) in both the hFOB/ER cell lines, certain responses to E or TAM (including alkaline phosphatase, IL‐6 and IL‐11 production) are more pronounced at the late mineralization stage of differentiation compared to earlier stages, (2) E exerted a greater regulation of bone nodule formation and matrix protein/cytokine production in the ERα cells than in ERβ cells, and (3) the regulated expression of select genes differed between the ERα and ERβ cells. TAM had no effect on nodule formation in either cell line and was a less potent regulator of gene/protein expression than E. Thus, both the ER isoform and the stage of differentiation appear to influence the response of osteoblast cells to E and TAM. J. Cell. Biochem. 83: 448–462, 2001.

Estrogen Receptor Isoform-Specific Induction of Progesterone Receptors in Human Osteoblasts†

Estrogen induction of progesterone receptor (PR) expression may be important to bone physiology because progesterone has been implicated in the control of bone formation and resorption. Although PR gene expression can be induced in osteoblasts by estrogen signaling through the estrogen receptor (ER) α isoform, it is unknown whether the ER‐β isoform is involved in this regulation. The effect of estrogen on PR expression was examined in human fetal osteoblast (hFOB) cell lines stably transfected with either ER‐α or ER‐β. Estrogen treatment of hFOB/ER‐α cells induced PR messenger RNA (mRNA) steady‐state levels after 24 h and protein levels after 48 h, as established by competitive reverse transcriptase‐polymerase chain reaction (RT‐PCR) and Western blotting. Interestingly, no induction of PR expression was observed in the hFOB/ER‐β cells during this period. However, PR mRNA was induced progressively after 48 h of treatment with estrogen with maximum levels achieved at 12 days posttreatment. ER protein also was increased after 12 days of treatment. Both A and B isoforms of PR (PRA and PRB) were induced by estrogen in the hFOB/ER‐α cells as well as much later in hFOB/ER‐β cells. The pure antiestrogen ICI 182,780 prevented PR induction by estrogen in both cell lines. An ER‐β‐selective antagonist R, R‐tetrahydrochrysene (THC) abolished the induction of PR mRNA in hFOB/ER‐β but not in hFOB/ER‐α cells, verifying that the response in the former cell line was ER‐β‐mediated. Transient cotransfection of hFOB cells with ER‐α or ER‐β together with either a human PRA or PRB promoter linked to a reporter plasmid revealed that although the PRB promoter was stimulated equally by estrogen activation of either ER isoform, PRA was activated preferentially by ER‐α. Together, these results show that although estrogen can up‐regulate endogenous PR gene expression in osteoblasts via both ER isoforms, ER‐α is the predominant inducer.

A Key Role for the Endothelium in NOD1 Mediated Vascular Inflammation: Comparison to TLR4 Responses

Understanding the mechanisms by which pathogens induce vascular inflammation and dysfunction may reveal novel therapeutic targets in sepsis and related conditions. The intracellular receptor NOD1 recognises peptidoglycan which features in the cell wall of Gram negative and some Gram positive bacteria. NOD1 engagement generates an inflammatory response via activation of NFκB and MAPK pathways. We have previously shown that stimulation of NOD1 directly activates blood vessels and causes experimental shock in vivo. In this study we have used an ex vivo vessel-organ culture model to characterise the relative contribution of the endothelium in the response of blood vessels to NOD1 agonists. In addition we present the novel finding that NOD1 directly activates human blood vessels. Using human cultured cells we confirm that endothelial cells respond more avidly to NOD1 agonists than vascular smooth muscle cells. Accordingly we have sought to pharmacologically differentiate NOD1 and TLR4 mediated signalling pathways in human endothelial cells, focussing on TAK1, NFκB and p38 MAPK. In addition we profile novel inhibitors of RIP2 and NOD1 itself, which specifically inhibit NOD1 ligand induced inflammatory signalling in the vasculature. This paper is the first to demonstrate activation of whole human artery by NOD1 stimulation and the relative importance of the endothelium in the sensing of NOD1 ligands by vessels. This data supports the potential utility of NOD1 and RIP2 as therapeutic targets in human disease where vascular inflammation is a clinical feature, such as in sepsis and septic shock.