85

Review

Biology of bone endothelial

cells

The fabric&m of living bone is a masterpiece of organization, where the microvasculature appears to play a major role. There is little oaderstanding of the role of bone endothelial cells in bone formation and their interactions with stromal and hematopoietic systems of bone tissue. The need for understanding the functional cooperation of these cells is, therefore, urgently felt. The endothelial cell has been traditionally regarded as important solely for its role in maintaining vascular integrity. Indeed, the basic functions performed by endothalial cells are similar in blood vessels of all sizes. Endothelial cells mast provide a nonthrombogenic surface to which platelets and other blood cells will fail toadCorrespondence to: Maria Luisa Brandi. Department of Clinical Physiopathology. University of Plw ewe, Vi&

Piemccini 6.50139 Florence, Italy.

0169.%#9~WisO3.50~

1990 Elsevier Science Publishers B.V. (Biomedical Division)

86 here. They must mediate the passage of nutrients and other solutes from the blood to tissues and maintain a patent lumen by growing as a single cell-layer tightly adherent to the basement membrane of the vessel wall. Besides acting as a selective permeability barrier and a blood compatible cootainer, endothelial cells are now recognized to have a variety of synthetic and metabolic capabilities. In fact, endothelial cells regulate vascular tone by synthe.sizing vasoactive agents [1,2]. Despite these functional similarities, there is reason to believe that there arc sionificant physiological differences among endothelial cells from diierent species and tissues. For example, bovine endothelial cells seem to grow more readily than those from other animal species. In addition, significant differences exist between the endothelial cells in the microvasculature and those in larger arteries and veins. ‘In vitro’ studies have shown that capillary endotheliil cells grow best when cultured on a substratum coated with basement membrane components, whde aortic and venous endothelial cells grow rapidly on uncoated plastic tissue culture dishes. Moreover, capillary endotbelial cells migrate in response to chemotactic factors released by tumor cells and mast cells; in contrast, aortic endotheliil cells do not [3,4]. Endothelial cells cloned from large vessels show a limited life-span in vitro [S], while capillary endothelial cells from different species have been maintained in long-term cultures (61. Finally, some disease states involve pathological changes in the microvasculature, while others preferentially affect large vessels. There is also considerable evidence that the capillary endothelial cells tbemselves differ from organ to organ. The first recognized difference resides in the ul?rastructure of the capillary endothelium in diverse sites. Electron microscopy has revealed that there are almost as many varieties of capillary endothelial cells as there are organs and tissues. The most obvious difference is in the continuity of capillary endothelial cells. Differences in intercellular endothelial connections form the distinction among three types of capillaries: continuous, discontinuous and fenestrated. In the brain and retina, where the capillaries participate in maintaining the blood brain barrier, adjacent endothelial cells are connected by continuous intercellular tight junctions. In capillaries of the liver, spleen and bone matrow, there are gaps between adjacent endotheliai cells, forminy discontinuous capillary endothelium. In humans, discontinuous endothelium lack Weibel-P&de bodies, where Von WiIlebrand’s factor is stored. A third type of capillary, termed fenestrated, has intracellular gaps within the cytoplasm of individual cells. Feoestrated cells are found in the capillaries of endocrine glands, intestinal vilti and kidney. Finally, lymphatic capillaries are characterized by extracellular filaments anchoring endotheiial cells to the perivascular connective tissue, thereby allowing the endothelium to maintain an open lumen when edema occurs. Recent evidence suggests that capillary endothetia are also functionally hetero. geneous. In the brain, microvascular andothelial cells synthesize and store neurotransmitter substances with vasoactive potency and exhibit specific enzymatic activities, such as glutamyltranspeptidase, whereas endothelial cells from other districts do not 171.Endothelial cells from the adrenal medulla can take up and deami“ate catecholamines by type-A monoamine oxidase [8]. Parathyroid endothelial cells are able to take up pamthyroid hormone in vitro; endothelia from other tissues

87 are not [9]. Capillary endothelial cells in adipose tissue may be involved in specific iipolyttc reactions [lo]. What is known about bone endothelium?

Bone

vasculature

The vasculature in bone is important in skeletal development and repair and may direct new bone formation by sewing as a scaffolding for osteobktsts. The developmental pattern of the vascular network seems to be an important component reflecting or directing Limbdevelopment (111.In early osteogenesis, the cartilage may produce an antiangiogenic factor inhibiting vascular penetration [12]. Vascularization is required before osteogenesis will occur, yet the reasons for thii dependence have not been fully elucidated. A combination of factors including adequate oxygen tension, compression forces, nutrients and growth or differentiation factors promote bone formation at the site where vascttlarization occurs. After vascular invasion, the hypertrophied cartilage core is degraded and replaced hy hone marrow and later by bone. Only cells which are situated near blood flow eapigaria give rise to bone tissue. Mineral formation is observed in the hnmediate vi&thy of vessels and seems to correlate with the onset of cartilage vascularization [13]. In osteoporotic bone tissue, as well as in aplastic anemia, mierovaseular defects are present in the sinusoidal compartment with a reduction of bone blood flow [14,15]. In onlay bone transfers, vascularired bone grafts retain their mass to a greater degree than nonvascular&d tissue [16]. In addition, electrical stimulation increases bone formation with a parallel increase ia the number of capillaries [17]. Bone endothelial cells, despite providing the basic endothelii functions, bave an essential role in the release of blood cells [18,19]. Moreover, the intimacy of endotlteIium-osteoblat contact has led to the theory that the endothelial cell itself is the osteoblast preeursor [Xl]. Finally, during vascular invasion of the hypertrophied cartilage core, endothelial cells of the advancing capillaries may have a direct or indhect role in resorption. Recently, aortic endothelial ceils have been shown to synthesize bone cell active mitogen(s) and, therefore, the endothelium may represent an important element in the genesis of bone fortnation [Z]. Despite the ituportanee of bone vasculature, previously the only available in vitro model was an endothelial cell population derived from rodent bone marrow [22]. The lack of other in vitro models may be explained by the extreme difficulty in investigating the vasetdature of a tissue as hard as hone. We investigated the possibility of developing in vitro ckmal cell populations of bovine bone endothelial cells. Only recently we succeeded in isolating bone cells as cultured clones and in identifying them as endothelial cells [23].

Bono endothelial cells

Most investigators acknowledge the difficulty of obtaining pure cuItures of bone endothelial cells. This is not surprising since bone fragments which serve as the source

88 for endothelial cells also contain a variety of stromal cells [24]. To obtain pure endothelial cell cultures, therefore, it is important to characterize the cells present in cultures of bone fragments. Ideally, cultured bone microvascular rndothelial cells should express general endothelial cell markers a:‘ we!! as more specialized properties of bune capillary endothelial cells. Primary cultures from fetal bovine sternum were developed after enzymatic digestion and mechanical dispersion. Cells were cultured and cloned in Coon’s modi-

Fig. 1. Morpholocof BBE-1cells.(A) Branchingor ‘rubaier’s!~.cts,~~.(II)

SpmutingceU(atrow).

x9

Bed Ham’s F12 containing 10% Nu-serum and 1% Ultroser-G (both are serum substitutes containing high concentrations of endothelial cell growth factors), calcium concentration 0.3 mM [23]. We have used the same medium to clone endothelial cells from bovine parathyroid tissue [9]. One clone, BBE-1, was chosen for analysis of morphology, growth characteristics and response to growth factors. To conclude that a cell is endothelial, rt must display typical endothelial morphologicai features, stain positively for factor VIII-related antigen and display characteristic mitogenic responses to fibroblast growth factors. BBE-1 cells displayed typica! endothelial morphology by light and electron microscopy. By light microscopy, at low density, they were small, triangular cells. At confluence, BBE-1 cells formed extensive imercellular connections and branching (Fig. 1A) and occasional sprouting (Fig. 1B). Branching, or ‘ttbular’ structures were shown by electron microscopy to be composed of parallel stacks of cells [23]. A three-dimensional gel may be naewed to see actual tube formation. Typical endothelial features of perinuclear granules, elongated mitcchoodtia. abundant rough endoplasmic reticulum and apparent vesicular shuttling were also noted [231. BBE-1 cells showed positive bnmunotluorescence for factor VIII-related an& gen. They exhibited evidence of senescence (nuclear and cellular enlargement) after 5-6 months in culture but retained branching morphology and positive factor VIII-related antigen staining throughout their lifespan of 8 months in continuous culture. The doubling time for early passage ceils was 48 h. Mitogeoic responses

were noted in response to endothelial cell growth factor alpha, ascorbic acid, insulin, insulin-like growth factor types I and 2, basic fibroblast growth factor and progesterone (Fig. 2). Inhibition of thymidine incorporation was noted in response to transforming growth factor beta and high dose heparin (loO,ug/ml). No mitcgenic response was seen after incubation with broad concentration ranges of calcitonin, epidermal growth factor, rat parathyroid hormone, dexamethasone, 1,25-dihydroxyvitamin D,, estradioi, testosterone, thrombin and transferrin [23]. Intracellular CAMP production increased in response to incubation with parathyroid hormone in BBE-1 cells but not in endotheiiil ceils from bovine pulmonary artery or parathyroid (Fig. 3). We know of no other report of parathyroid hormone responsiveness in other pure endothelial eel1 cultures. BBGl eelis had no detectable alkaline phosphatasc acticitj [23j. In addition, incubation of BBE-1 cells with parathyroid hormone or transforming growth factor beta for 2 weeks failed to induce alkaline uhosohatase activity furpubiiihed observations). Preliminary studthat BEE-1 cells (early and iate passage) produce abundant type 1 collagen and osteonectin and small amounts of fibronectin and thrombospondm under standard culture conditions (Dr Pamela Robey, personal communication).

It is remarkable that the importance of bone endotheiium in bone formation and resorption was postulated many years ago [20,2.5],but these observations are rarely mentioned in current literature. A schematic of the different locations of bone mi-

Fig.3. latraceltutarCAMP productionin responseto rat parathyroidhormonein endothelirdalls

fmm bovinepulmanary zu&ry, parathymid and bone @BE-l). CcUswere incubatedfor 10 min with para-

thyroid hormone and CAMP determined by standardmethodr utilizing an automated RIA IW

for CAMP

91

1. OSTHxl OF COMFWT SONE

5. SPROUTING cAPI_ INVAOING EFlPWSEAL PLATE

92

crovascular cells is depicted in Fig. 4. The functional connections among endothelial cel!s, osteoblasts and osteoclasts arc evident. In osteon formation, the gradual filling-in process that converts cancellous bone into compact bone creates a number of narrow canals that are lined with osteogenic cells. These canals enclose vessels that were formerly present in soft tissue spaces in the cancellow network (Fig. 4.1). Also, on other bone surfaces (the deeper layers of the periosteum and endosteum that line the internal surfaces of all cavities within bone), the osteogenic cells are intimately associated with blood vessels (Fig. 4.2). Endochondral and intramembranow ossification proceed in association with capillaries (Fig. 4.2,4.3). In the fcrmation of ossification centers in long bones, mineralization occurs only when arterioles penetrate the periosteum, invading cartilege (Fig. 4.2). In the epiphyseal plate, ossification occurs when metaphyseal capillary sprouts appear (Fig. 4.4). In the intramembranous ossification model, the center of osteogenesis develops in association with capillaries that grow into the mesenchyme (Fig. 4.3). The mesenchyma1 cells, characterized by a stellate appearance and pale cytoplasm, pass imperceptibly through the osteogenic cell stage, becoming more rounded and basophilic, with thicker interconnecting processes. An intimate functional relationship also exists between the sproutiog capillaries invading the epiphyseal plate and osteoclastic and osteoblastic cells (Fig. 4.5). In fact, osteoclasts are typically located on the tip of the spmuting capillary, in a strategic position to resorb calcified cartilage, while the osteoblasts line the terminal wall of the capillary, like the rearguard ready to generate the organic matrix of bone. A frequent paratrabecular continuity of the sinusoid endothelium and the osteoblastic seams, together with the possible direct endosteal production of some sinusoidal compartment [26] represent convincing evidence of a relationship between endothelial cells and osteoblasts. Our in vitro ccl1model represents a tool to address some of these questions. The binding of a PTH-sensitive adenylate cyclase in endothelial cells from bone but not in cells from other districts is provocative. Might this represent a genetic relationship between bone endothelial cells and osteoblasts? This possibility needs to be analyzed in detail, considering also the existence of a family of perivascular cells (pericytes, smooth muscle cells and fibroblasts) that may play an integrating role in this context.

Future perspectives The endothelium influences the growth and differentiation of parenchymal tissues. Little is known about the interactions between endothelial cells and other cells in bone. The nature of such interactions is an emerging subject which promises tc. be of great interest. The best available model for in vitro studies of bone endothelium is represented by the BBE-1 cells. This system allows direct testing of possible functional interactions with cells of osteoblastic and osteodastic lineages. Preliminary studies with Boyden chambers support the hypothesis of a stimulatory effect of bone endothelial cells on pre-osteoclastic cell [27] migration (unpublished data). The effect of bone endothe!ial cells on osteoclast maturation and differentiation

93 could be similarly evaluated. These studies will make it possible to isolate the cell surface molecules important in controlling os!eodast precursor homing in bone tissue. Preliminary experiments have demonstrated that BBE-I cells foist a distinctive stellate pattern when co-cultured with osteoblast-like MC3T3 cells and conditioned media from BBE-1 cells is mitogenic for these osteoblast-like cells (unpublished observations). The finding of CAMP production in response to parathyroid hormone in BBE-1 cells is provocative and allows speculation about possible relationships between endothelial cells and osteoblasts. However, to rigorously prove a commonorigin for osteoblastic and endothelial cells, one wonld have to demonstrate the differentiation of one cell type into the other. A lack of such transition in determined cell lineages, however, would not totally exclude the possibility of a common ancestor. In fact, interaction with other bone tissue elements might be necessary to create a favorable microenvironment for induction. Moreover, in our present state of knowledge, it is not possible to distinguish between bone endothelial cells deriving from different compact tissue areas or marrow niches. Heterogeneity in the pattern of differentiation of bone endothelial cells from varied vessels may exist. Simple (two components) or multiple (three or more components) co-culture sy% terns may make it possible to recreate the complexity of the bone microenvironment and therefore to optimize suitable in vitro conditions for osteodifferentiation. Finally, more efforts are needed to select endothelial cell populations from bone tissues. With these tools in our hands we may possibly understand the importance of the endothelium in bone cell physiology in normal and pathological conditions.

We would like to thank Dr Gerald Aurbach for his continuing support and valuable contributions in these studies.

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