Thursday, November 5, 2009

Epiphyseal Plate Vascularization

Epiphyseal plate vascularization refers to the post hypertrophic stage of the growth plate where the growth plate is invaded by blood vessels before the ossification zone forms.

Matrix vesicles: structure, composition, formation and function in calcification.

"Matrix vesicles (MVs) induce calcification during endochondral bone formation. MV proteins, enzymes, receptors, transporters, regulators, lipids and electrolytes are detailed. MV formation is considered from both structural and biochemical perspectives. Confocal imaging of Ca(2+) and H(+) were used to depict how living chondrocytes form MVs. coordinated mitochondrial Ca(2+) and Pi metabolism produce MVs containing a nucleational complex (NC) of amorphous calcium phosphate, phosphatidylserine and annexin A5--all critical to the mechanism of mineral nucleation. Reconstitution of the NC and modeling with unilamellar vesicles reveal how the NC transforms into octacalcium phosphate, regulated by Mg(2+), Zn(2+) and annexin A5. Extravasation of intravesicular mineral is mediated by phospholipases and tissue-nonspecific alkaline phosphatase (TNAP). In the extravesicular matrix, hydroxyapatite crystal propagation is enhanced by cartilage collagens and TNAP, which destroys inhibitory PPi, and by metalloproteases that degrade proteoglycans."

"Endochondral calcification is a primary, but provisional process; its end product, calcified cartilage, is more heavily calcified, but is mechanically weaker than true bone. It is destined to be largely replaced by cancellous bone. Endochondral calcification is mediated by growth plate chondrocytes; it is a rapid de novo process that begins with Ca2+ and Pi ions in solution. It involves overcoming of a sizeable nucleation barrier that requires the direct mediation of cellular metabolic activity. It utilizes extracellular vesicles and ends with formation of large amounts of microcrystalline, Ca2+-deficient, acid-phosphate-rich apatite deposits embedded in a proteoglycan and cartilage-specific collage-rich matrix."

"Type VI collagen receptors on the chondrocyte surface (NG2-proteoglycan) act as transducers to mitochondria within the cells, affecting their permeation transition pore for Ca2+" LSJL upregulates Col6a1,Col6a2, and Col6a3.  And downregulates another form of Col6a2.  Col6 is involved in matrix vesicle formation.

"Type X collagen had a small inhibitory effect on the onset and rate of mineral formation; however it significantly extended the length of the "rapid formation" period, thereby altering the kinetics of the overall amount of mineral formation"

Anxa5, 6, 1, 11, 4, and 2 are associated with MVs.

"AnxA5 greatly accelerates the nucleational activity of the acidic phospholipid-Ca2+-Pi complexes present in the nucleational core that triggers de novo calcium phosphate mineral formation in MVs. AnxA5 specifically overcomes the inhibitory effects of Mg2+ that otherwise greatly slow the induction of mineral formation"

"Expression of AnxA6 in cells reduces cell proliferation"

"AnxA2 has been implicated in the detachment of membrane plaques from actin filaments, leading to the formation of cell-surface blebs"

"NPP1 accounts for nearly all the ecto-pyrophosphatase activity of chondrocytes and the release of PPi is thought to provide a means for regulating apatitic mineral formation"

"phospholipid scramblase activity in growth plate chondrocytes [is associated] with TNAP-containing lipid rafts during MV formation"

"In the maturing growth plate chondrocytes, the presence of TRPV4 very likely has an important function in the major osmotic swelling that occurs upon hypertrophy when cell volume expands over 9-fold going from the proliferating to the hypertrophic state. Also of interest are CLIC4 - the Channel 4 intracellular Cl- transporter; this channel protein transports Cl- in exchange for HCO3-, and appears to be important in buffering the internal pH of MVs. The third is VDAC-2, - the Channel 2 voltage-dependent anion-selective channel present in the mitochondrial outer membrane (312-313). This channel may play a part in the mitochondrial permeability transition pore that becomes activated prior to formation of MVs by hypertrophic chondrocytes"

SLC's involved in MV's:

"SLC 16A1{down} [is] a transporter known to transport lactate, pyruvate"

"neutral amino acids (SLC38A3), alanine/serine/cysteine/threonine (SLC1A4){up}, organic cations, e.g. carnitine (SLC22A16), glycine (SLC6A9), Na+-dependent citrate (SLC13A5), Na+-independent cationic amino acids (SLC7A3){down}, and equilibration of nucleosides (SLC29A1). "

"two Zn2+ transporters - SLC39A14 and SLC39A8."

"Pi transporters use an inwardly directed electrochemical Na+ current to support Pi influx. This Na+ gradient, produced by the action of a Na+/K+ ATPase in the plasma membrane, carries Pi into living cells. However in MVs, Na+ levels are as high as that of the extravesicular fluid; thus there is no Na+ gradient to drive Pi uptake."

"Substitution of Li+ or K+ for Na+ had minimal effect on Pi uptake; N-methyl D-glucamine (NMG+) was totally inhibitory, whereas choline+ was clearly stimulatory"

"syntenin binds to syndecan, which in turn binds to type VI collagen in the extracellular matrix, leaving MVs attached at the points of contact"

" phalloidin, an agent that stabilizes actin microfilaments, significantly decreased MV formation. Another drug, colchicine, which destabilizes microtubules, also stimulated the release of MVs."

"both microtubules and microfilaments were involved in the formation of cell processes from which MV were shown to derive"

"accumulated mitochondrial Ca2+ becomes subsequently transferred to MV during growth plate development."

"Accumulation of excessive Ca2+ is known to inhibit mitochondrial respiration "

"Growth plate cartilage has been shown to possess unusually high levels of both cytosolic (20-25 mM) and extracellular (2.2-2.5 mM) Pi"

"cultured tibial growth plate chondrocytes to even modestly elevated levels (3 mM) of extracellular Pi induces 'apoptotic' cell death within 48 h."

"Mitochondrial uptake of Pi is largely accomplished by PiC, an H+/Pi symporter (or Pi/OH- antiporter). PiC is an integral membrane protein, a homodimer that resides in the inner mitochondrial membrane that translocates or exchanges Pi from the cytosol into the mitochondrial matrix"

"PiC is Na+-independent and is responsible for the rapid import of Pi used for ATP synthesis."

"During MV formation, PS-CPLXs can readily form at pH 6.7, conditions like that present in the perimeter of growth plate chondrocytes. In contrast, the ACP component of the nucleational core cannot form at this pH and can occur only after the vesicles are released into the extracellular fluid where the pH (7.6) favors ACP formation.  A key requirement in the formation of PS-CPLX is that excess Pi must be present[which occurs during MV formation] with the lipid before introduction of Ca2+"

"[factors] influencing MV nucleation include: a) the inhibitory effects of Mg2+, Zn2+, and PPi, b) the aforementioned synergistic effects of the MV annexins and PS, and c) the inhibitory effects of PE and other lipid components of the MV membrane."

"MVs are formed by growth plate cells whose cytosolic levels of Mg2+ are much higher than those of Ca2+ . Thus, all PS-Ca2+-Pi complexes formed in vivo incorporate both Mg2+ and Zn2+; in fact analyses of the complexes present in avian MVs have shown that Mg2+ is almost as abundant as Ca2+. Our recent studies have revealed that this level of Mg2+ is profoundly inhibitory to the nucleational activity of PS-Ca2+-Pi complexes, greatly reducing their rate . This inhibitory effect is physiologically important because as noted above rapidly growing species like broiler-strain chickens have such fast longitudinal growth that speedy nucleation is essential for mineral formation to keep pace."

"levels of extravesicular Zn2+ as low as 5 microM are sufficient to totally block MV mineral formation"

" MVs bind tightly to type II and X collagens present in growth plate cartilage"

"during growth plate development the chondrocytes initially require Pi for synthesis of DNA, RNA and nucleotides essential for proliferation. Upon termination of cell division, the nucleotides begin to be degraded; 31P-NMR and chemical analyses reveal that Pi builds up in the cytoplasm. Simultaneously, in situ confocal imaging reveals that the chondrocytes acquire and process Ca2+ at an accelerated pace until in the early hypertrophic zone this climaxes with the cells exfoliating from their plasma membrane the accumulated Ca2+ and Pi as ion-loaded vesicles. This mineral phase, present as a nucleational core within the vesicle lumen, is primarily noncrystalline calcium phosphates complexed with both PS and the annexins, which facilitate the induction of crystalline mineral formation. When MVs are formed by the cells they contain not only Ca2+ and Pi, but also other electrolytes that significantly affect the kinetics of mineral formation; these include Mg2+, Zn2+, and PPi, as well as HCO3-. Production of mineralization-competent MVs enables mineral formation to keep pace with cellular proliferation, preventing development of rickets or osteomalacia. Thus, endochondral bone formatioin involves the provisional calcification of the cartilaginous matrix by chondrocytes - i.e. the formation of calcified cartilage - a more heavily calcified, but mechanically weaker temporary scaffolding that maintains mechanical stability until the truly robust type I collagen-hydroxyapatite composite, bone, is generated by osteoblasts."

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