Patricia A. Veno

Cytostatic properties of some angiotensin I converting enzyme inhibitors and of angiotensin II type I receptor antagonists.

Angiotensin converting enzyme (ACE) inhibitors and angiotensin II (AII) type 1 receptor antagonists have strong cytostatic properties on in vitro cultures of many normal and neoplastic cells. They are effective, in particular, in reducing the growth of human lung fibroblasts, renal canine epithelial cells, bovine adrenal endothelial cells, simian T lymphocytes, and of neoplastic cell lines derived from human neuroblastomas, a ductal pancreatic carcinoma of the Syrian hamsters, human salivary glands adenocarcinomas, and two lines of human breast adenocarcinomas. ACE inhibitors are also effective in protecting lungs, kidneys and bladders from the development of nephropathy, pneumopathy, cystitis, and eventually fibrosis in different models of organ-induced damage such as exposure to radiation, chronic hypoxia, administration of the alkaloid monocrotaline or bladder ligation. ACE inhibitors and AII type 1 receptor antagonists are also effective in reducing excessive vascular neoformation in a model of injury to the cornea of rats and rabbits, and in controlling the excessive angiogenesis observed in the Solt-Farber model of experimentally induced hepatoma, in methylcholantrene or radiation-induced fibrosarcomas, in radiation-induced squamous cell carcinomas and in the MA-16 viral-induced mammary carcinoma of the mouse. Captopril was, in addition, effective in controlling tumor growth in a case of Kaposis sarcoma in humans. The inhibition of AII synthesis and/or its blockade by AII receptors is likely to be an important mechanism for this cytostatic action. The mitogenic effect of AII is well established and a reduction of AII synthesis may well explain cell and neoplasm delayed growth. Moreover, AII regulates and enhances the activity of several growth factors including transforming growth factor B (TGFB) and smooth muscle actin (SMA); and many of these factors are reduced in tissues of animals treated with ACE inhibitors and AII type 1 receptor antagonists. These processes seem to be particularly relevant in the control of fibroblast growth and in the control of the ensuing fibrosis. The ACE inhibitors containing a sulphydril (SH) or thiol radical in their moiety (Captopril and CL242817) seemed to be more effective in controlling fibrosis and the growth of some neoplastic cells than those ACE inhibitors without this thiol radical in their structure, even if the second group of these drugs show in vitro a stronger inhibitory effect on converting enzyme activity. Pharmacologically it is known that ACE inhibitors containing a thiol radical also have antioxidant properties and they are efficient in controlling metalloproteinase action. However, although these additional properties are pharmacologically relevant, the blockade of AII synthesis plays an essential role in the cytostatic activity of these two categories of drugs. These observations underline that in addition to the beneficial effect of these drugs on the cardiovascular system, new potential applications are opening for their wider deployment.

Prevention of radiation-induced nephropathy and fibrosis in a model of bone marrow transplant by an angiotensin II receptor blocker.

Nephropathy, interstitial pneumopathy, and renal and lung fibrosis are major complications of bone marrow transplantation (BMT). This study evaluated the antifibrotic property of an angiotensin II (A2) type-1 receptor blocker (L-159,809) and compared it with those of Captopril and Enalapril, two angiotensin-converting enzyme (ACE) inhibitors, in a rat model of BMT. Male WAG/Rij/MCW rats received a preparative regimen of 60 mg/kg body wt of cytoxan (i.p., Days 9 and 8) and 18.5 Gy of total body irradiation (TBI) in six twice daily fractions (Days 2, 1, and 0) followed immediately (Day 0) by BMT. Modifiers were given in drinking water from Day 10 until autopsy, 8 weeks after BMT. Rats treated with TBI plus cytoxan alone developed severe nephropathy. Trichrome staining showed marked collagen deposition in glomeruli, renal interstitium, and renal arteries and arterioles (especially in their adventitia). Collagen deposition and renal damage were markedly reduced by the three modifiers. Of the three, L-158,809-treated rats had slightly thinner vessels and slightly less collagen than nonirradiated normal controls. The study shows the effectiveness of these drugs in the protection of the renal parenchyma from the development of radiation-induced fibrosis. It also indicates a role for angiotensin II in the modulation of collagen synthesis.

Identification of osteocyte-selective proteins

Since little is known regarding osteocytes, cells embedded within the mineralized bone matrix, a proteomics approach was used to discover proteins more highly expressed in osteocytes than in osteoblasts to determine osteocyte‐specific function. Two proteomic profiles obtained by two different proteomic approaches using total cell lysates from the osteocyte cell line MLO‐Y4 and the osteoblast cell line MC3T3 revealed unique differences. Three protein clusters, one related to glycolysis (Phosphoglycerate kinase 1, fructose‐bisphosphate aldolase A, hypoxia up‐regulated 1 [ORP150], triosephosphate isomerase), one to protein folding (Mitochondrial Stress‐70 protein, ORP150, Endoplasmin), and one to actin cytoskeleton regulation (Macrophage‐capping protein [CapG], destrin, forms of lamin A and vimentin) were identified. Higher protein expression of ORP‐150, Cap G, and destrin in MLO‐Y4 cells compared with MC3T3 cells was validated by gene expression, Western blotting, and in vivo expression. These proteins were shown to be selective in osteocytes in vivo using immuno‐staining of mouse ulnae. Destrin was most highly expressed in embedding osteoid osteocytes, GapG in embedded osteocytes, and ORP150 in deeply embedded osteocytes. In summary, the proteomic approach has yielded important information regarding molecular mechanisms used by osteocytes for embedding in matrix, the formation of dendritic processes, and protection within a hypoxic environment.

Time Lapse Imaging Techniques for Comparison of Mineralization Dynamics in Primary Murine Osteoblasts and the Late Osteoblast/Early Osteocyte-Like Cell Line MLO-A5

Mineralization of bone matrix and osteocyte differentiation occur simultaneously and appear interrelated both spatially and temporally. Although these are dynamic events, their study has been limited to using static imaging approaches, either alone or in combination with chemical and biochemical analysis and/or genetic manipulation. Here we describe the application of live cell imaging techniques to study mineralization dynamics in primary osteoblast cultures compared to a late osteoblast/early osteocyte-like cell line, MLO-A5. Mineral deposition was monitored using alizarin red as a vital stain for calcium. To monitor differentiation into an osteocyte-like phenotype, the calvarial cells were isolated from transgenic mice expressing green fluorescent protein (GFP) driven by an 8-kb dentin matrix protein-1 (Dmp1) promoter that gives osteocyte-selective expression. Time lapse imaging showed that there was a lag phase of 15–20 h after β-glycerophosphate addition, followed by mineral deposition that was rapid in primary osteoblast cultures but more gradual in MLO-A5 cultures. In primary osteoblast cultures, mineral was deposited exclusively in association with clusters of cells expressing Dmp1-GFP, suggesting that they were already differentiating into osteocyte-like cells. In MLO-A5 cells, the first indication of mineralization was the appearance of punctate areas of alizarin red fluorescence of 4–7 μm in diameter, followed by mineral deposition throughout the culture in association with collagen fibrils. A high amount of cell motility was observed within mineralizing nodules and in mineralizing MLO-A5 cultures. These studies provide a novel approach for analyzing mineralization kinetics that will enable us to dissect in a time-specific manner the essential players in the mineralization process.

Association of Specific Proteolytic Processing of Bone Sialoprotein and Bone Acidic Glycoprotein-75 with Mineralization within Biomineralization Foci

Mineral crystal nucleation in UMR 106-01 osteoblastic cultures occurs within 15–25-μm extracellular vesicle-containing biomineralization foci (BMF) structures. We show here that BAG-75 and BSP, biomarkers for these foci, are specifically enriched in laser capture microscope-isolated mineralized BMF as compared with the total cell layer. Unexpectedly, fragments of each protein (45–50 kDa in apparent size) were also enriched within captured BMF. When a series of inhibitors against different protease classes were screened, serine protease inhibitor 4-(2-aminoethyl)benzenesulfonylfluoride HCl (AEBSF) was the only one that completely blocked mineral nucleation within BMF in UMR cultures. AEBSF appeared to act on an osteoblast-derived protease at a late differentiation stage in this culture model just prior to mineral deposition. Similarly, mineralization of bone nodules in primary mouse calvarial osteoblastic cultures was completely blocked by AEBSF. Cleavage of BAG-75 and BSP was also inhibited at the minimum dosage of AEBSF sufficient to completely block mineralization of BMF. Two-dimensional SDS-PAGE comparisons of AEBSF-treated and untreated UMR cultures showed that fragmentation/activation of a limited number of other mineralization-related proteins was also blocked. Taken together, our results indicate for the first time that cleavage of BAG-75 and BSP by an AEBSF-sensitive, osteoblast-derived serine protease is associated with mineral crystal nucleation in BMF and suggest that such proteolytic events are a permissive step for mineralization to proceed.

Novel approaches for two and three dimensional multiplexed imaging of osteocytes.

Although osteocytes have historically been viewed as quiescent cells, it is now clear that they are highly active cells in bone and play key regulatory roles in diverse skeletal functions, including mechanotransduction, phosphate homeostasis and regulation of osteoblast and osteoclast activity. Three dimensional imaging of embedded osteocytes and their dendritic connections within intact bone specimens can be quite challenging and many of the currently available methods are actually imaging the lacunocanalicular network rather than the osteocytes themselves. With the explosion of interest in the field of osteocyte biology, there is an increased need for reliable ways to image these cells in live and fixed bone specimens. Here we report the development of reproducible methods for 2D and 3D imaging of osteocytes in situ using multiplexed imaging approaches in which the osteocyte cell membrane, nucleus, cytoskeleton and extracellular matrix can be imaged simultaneously in various combinations. We also present a new transgenic mouse line expressing a membrane targeted-GFP variant selectively in osteocytes as a novel tool for in situ imaging of osteocytes and their dendrites in fixed or living bone specimens. These methods have been multiplexed with a novel method for labeling of the lacunocanalicular network using fixable dextran, which enables aspects of the osteocyte cell structure and lacunocanalicular system to be simultaneously imaged. The application of these comprehensive approaches for imaging of osteocytes in situ should advance research into osteocyte biology and function in health and disease.

Parathyroid Hormone Induces Bone Cell Motility and Loss of Mature Osteocyte Phenotype through L-Calcium Channel Dependent and Independent Mechanisms

Parathyroid Hormone (PTH) can exert both anabolic and catabolic effects on the skeleton, potentially through expression of the PTH type1 receptor (PTH1R), which is highly expressed in osteocytes. To determine the cellular and molecular mechanisms responsible, we examined the effects of PTH on osteoblast to osteocyte differentiation using primary osteocytes and the IDG-SW3 murine cell line, which differentiate from osteoblast to osteocyte-like cells in vitro and express GFP under control of the dentin matrix 1 (Dmp1) promoter. PTH treatment resulted in an increase in some osteoblast and early osteocyte markers and a decrease in mature osteocyte marker expression. The gene expression profile of PTH-treated Day 28 IDG-SW3 cells was similar to PTH treated primary osteocytes. PTH treatment induced striking changes in the morphology of the Dmp1-GFP positive cells in IDG-SW3 cultures and primary cells from Dmp1-GFP transgenic mice. The cells changed from a more dendritic to an elongated morphology and showed increased cell motility. E11/gp38 has been shown to be important for cell migration, however, deletion of the E11/gp38/podoplanin gene had no effect on PTH-induced motility. The effects of PTH on motility were reproduced using cAMP, but not with protein kinase A (PKA), exchange proteins activated by cAMP (Epac), protein kinase C (PKC) or phosphatidylinositol-4,5-bisphosphonate 3-kinase (Pi3K) agonists nor were they blocked by their antagonists. However, the effects of PTH were mediated through calcium signaling, specifically through L-type channels normally expressed in osteoblasts but decreased in osteocytes. PTH was shown to increase expression of this channel, but decrease the T-type channel that is normally more highly expressed in osteocytes. Inhibition of L-type calcium channel activity attenuated the effects of PTH on cell morphology and motility but did not prevent the downregulation of mature osteocyte marker expression. Taken together, these results show that PTH induces loss of the mature osteocyte phenotype and promotes the motility of these cells. These two effects are mediated through different mechanisms. The loss of phenotype effect is independent and the cell motility effect is dependent on calcium signaling.

Hemorrhage activates proopiomelanocortin neurons in the rat hypothalamus

Severe blood loss lowers arterial pressure through a central mechanism that is thought to include opioid neurons. In this study, we investigated whether hemorrhage activates proopiomelanocortin (POMC) neurons by measuring Fos immunoreactivity and POMC mRNA levels in the medial basal hypothalamus. Hemorrhage (2.2 ml/100 g body weight over 20 min) increased the number of Fos immunoreactive neurons throughout the rostral-caudal extent of the arcuate nucleus, the retrochiasmatic area and the peri-arcuate region lateral to the arcuate nucleus where POMC neurons are located. Double label immunohistochemistry revealed that hemorrhage increased Fos expression by beta-endorphin immunoreactive neurons significantly. The proportion of beta-endorphin immunoreactive neurons that expressed Fos immunoreactivity increased approximately four-fold, from 11.7+/-1.4% in sham-operated control animals to 42.0+/-5.2% in hemorrhaged animals. Hemorrhage also increased POMC mRNA levels in the medial basal hypothalamus significantly, consistent with the hypothesis that blood loss activates POMC neurons. To test whether activation of arcuate neurons contributes to the fall in arterial pressure evoked by hemorrhage, we inhibited neuronal activity in the caudal arcuate nucleus by microinjecting the local anesthetic lidocaine (2%; 0.1 or 0.3 microl) bilaterally 2 min before hemorrhage was initiated. Lidocaine injection inhibited hemorrhagic hypotension and bradycardia significantly although it did not influence arterial pressure or heart rate in non-hemorrhaged rats. These results demonstrate that hemorrhage activates POMC neurons and provide evidence that activation of neurons in the arcuate nucleus plays an important role in the hemodynamic response to hemorrhage.

Live Imaging of Type I Collagen Assembly Dynamics in Osteoblasts Stably Expressing GFP and mCherry-Tagged Collagen Constructs: LIVE IMAGING OF TYPE I COLLAGEN ASSEMBLY

Type I collagen is the most abundant extracellular matrix protein in bone and other connective tissues and plays key roles in normal and pathological bone formation as well as in connective tissue disorders and fibrosis. Although much is known about the collagen biosynthetic pathway and its regulatory steps, the mechanisms by which it is assembled extracellularly are less clear. We have generated GFPtpz and mCherry‐tagged collagen fusion constructs for live imaging of type I collagen assembly by replacing the α2(I)‐procollagen N‐terminal propeptide with GFPtpz or mCherry. These novel imaging probes were stably transfected into MLO‐A5 osteoblast‐like cells and fibronectin‐null mouse embryonic fibroblasts (FN‐null‐MEFs) and used for imaging type I collagen assembly dynamics and its dependence on fibronectin. Both fusion proteins co‐precipitated with α1(I)‐collagen and remained intracellular without ascorbate but were assembled into α1(I) collagen‐containing extracellular fibrils in the presence of ascorbate. Immunogold‐EM confirmed their ultrastuctural localization in banded collagen fibrils. Live cell imaging in stably transfected MLO‐A5 cells revealed the highly dynamic nature of collagen assembly and showed that during assembly the fibril networks are continually stretched and contracted due to the underlying cell motion. We also observed that cell‐generated forces can physically reshape the collagen fibrils. Using co‐cultures of mCherry‐ and GFPtpz‐collagen expressing cells, we show that multiple cells contribute collagen to form collagen fiber bundles. Immuno‐EM further showed that individual collagen fibrils can receive contributions of collagen from more than one cell. Live cell imaging in FN‐null‐MEFs expressing GFPtpz‐collagen showed that collagen assembly was both dependent upon and dynamically integrated with fibronectin assembly. These GFP‐collagen fusion constructs provide a powerful tool for imaging collagen in living cells and have revealed novel and fundamental insights into the dynamic mechanisms for the extracellular assembly of collagen.