Achieve height increase by modifying MMPs?

MMP(or matrix metalloproteinase) are one of the compounds affected by F-spondin and thus could potentially have height decreasing effects.  Zinc stimulates MMP so if MMPs have some height increasing effects then Zinc could help increase height there are also some MMP inhibitors available.  MMP-9 along with VEGF and osteoclasts are involed in chondrocyte ossification.  What are matrix metalloproteinase and how do they affect height? 

Involvement of matrix metalloproteinases in the growth plate response to physiological mechanical load. 

"Enzymes from the matrix metalloproteinase (MMP) family play a crucial role in growth-plate vascularization and ossification via proteolytic cleavage and remodeling of the extracellular matrix. Their regulation in the growth plate is crucial for normal matrix assembly. Endochondral ossification, which takes place at the growth plates, is influenced by mechanical loading. Using an in vivo avian model for mechanical loading, we have found increased blood penetration into the growth plates of loaded chicks. The purpose of this work was to study the involvement of MMP-2, -3, -9, -13, and -16 in the growth plate's response to loading and in the catch-up growth resulting from load release. We found that mechanical loading, as well as release from load, upregulated MMP-2{up in LSJL}, -9, and -13 expressions. MMP-3, associated with cartilage injuries, and its associated protein connective tissue growth factor (CTGF), were downregulated by the load. However, after release from load, MMP-3{up in LSJL} was upregulated and CTGF levels were elevated and caught up with the control. MMP-3 and CTGF were also downregulated after 60 min of mechanical stretching in vitro." 

"In human vein cells, stationary strain significantly increased MMP-2, whereas cyclic strain decreased it. In those same cells, MMP-9 increased in response to stationary strain, but exhibited no response to cyclic strain. Static compression loading on rat caudal vertebrae elevated the expression of MMP-3, but not MMP-13; in bovine synovial cells, MMP-3, but not MMP-1, increased after cyclic tensile strain"

"Connective tissue growth factor (CTGF/CCN2) is regulated by MMP-3 in two ways:
1) it cleaves CTGF protein into two ∼20-kDa fragments that are thought to have distinct functions;
2) it acts intracellularly to enhance CTGF transcription by directly binding to the CTGF promoter. MMP-3 [upregulates] the expression of CTGF in the human chondrocyte cell line HCS-2/8. CTGF promotes proliferation, maturation, and hypertrophy of growth-plate chondrocytes during endochondral ossification. It acts as a central driver of cartilage and bone regeneration. CTGF level is increased in HCS-2/8 cells after exposure to cyclic mechanical force, but is reduced after exposure to cyclic tension force"

"A reduction in growth-plate width, together with an increased number of vessels penetrating the growth plate from the metaphysic, was observed [in response to growth plate stretching]"

"Together with the increase in blood penetration, a quantitative increase in the expression of MMP-2, [-3], -9, and -13 was observed in the [loading] released chicks"<-LSJL is more consistent with release from load.

"CTGF is expressed in the prehypertrophic zone of the growth plate. It regulates chondrocyte proliferation and differentiation by forming a complex with BMP-2. In postfracture regenerating cartilage, its expression is enhanced in the hypertrophic and proliferative chondrocytes, and it is regulated by MMP-3 "

"the process of catch-up growth may resemble that of wound healing."<-Maybe LSJL operates similarly to catch up growth?

Now the increase in MMP-9 could be a result of increased chondrocyte hypertrophy and not the mechanical loading causing premature ossification of the chondrocytes. 

Effects of bisphosphonate on the endochondral bone formation of the mandibular condyle. 

"This study examined the effects of bisphosphonate on the growth of the condylar cartilage. Alendronate (3.5 mg/kg/week) was administered to postnatal day 1 SD rats for 7 and 10 days. The anteroposterior diameter of the developing condyle was unaffected by the alendronate treatment for 7 days. The total thickness of the cartilage layers was also unaffected by the treatment for 7 days. In particular, there was no change in the thickness of the perichondrium and reserve cell layer at the measured condylar regions. The thickness of the proliferating cell layer was reduced significantly, whereas the thickness of hypertrophied cartilage layer was increased. The number of chondroclasts engaged in hypertrophied cartilage resorption was reduced significantly by the alendronate treatment. The level of MMP-9 expression was reduced at both the transcription and translation levels by the alendronate treatment for 7 and 10 days. alendronate (>3.5 mg/kg/week) inhibits the longitudinal growth of the mandibular condyle by inhibiting chondrocyte proliferation and the resorption of hypertrophied cartilage for ossification." 

So MMP-9 may not only have the possibly negative effect of resorping hypertrophic cartilage, it may also promote chondrocyte proliferation. 

Localization of tartrate-resistant acid phosphatase (TRAP), membrane type-1 matrix metalloproteinases (MT1-MMP) and macrophages during early endochondral bone formation. 

"Endochondral bone formation, the process by which most parts of our skeleton evolve, leads to the establishment of the diaphyseal primary (POC) and epiphyseal secondary ossification centre (SOC) in long bones. An essential event for the development of the SOC is the early generation of vascularized cartilage canals that requires the proteolytic cleavage of the cartilaginous matrix[could development of new vascularized cartilage canals lead to new growth plates?]. This in turn will allow the canals to grow into the epiphysis. In the present study we therefore initially investigated which enzymes and types of cells are involved in this process. We have chosen the mouse as an animal model and focused our studies on the distal part of the femur during early stages after birth. The formation of the cartilage canals was promoted by tartrate-resistant acid phosphatase (TRAP) and membrane type-1 matrix metalloproteinases (MT1-MMP). In addition, macrophages and cells containing numerous lysosomes contributed to the establishment of the canals and enabled their further advancement into the epiphysis. As development continued, the SOC was formed, and in mice aged 10 days a distinct layer of type I collagen (= osteoid) was laid down onto the cartilage scaffold. The events leading to the establishment of the SOC were compared with those of the POC. Basically these processes were quite similar, and in both ossification centers, TRAP-positive chondroclasts resorbed the cartilage matrix. However, occasionally co-expression of TRAP and MT1-MMP was noted in a small subpopulation of this cell type. Furthermore, numerous osteoblasts expressed MT1-MMP from the start of endochondral ossification, whereas others did not. In osteocytogenesis, MT1-MMP has been shown to be critical for the establishment of the cytoplasmic processes mediating the communication between osteocytes and bone-lining cells.  Not all osteoblasts transform into osteocytes. MT1-MMP is needed at the very beginning of osteocytogenesis and may determine whether an osteoblast further differentiates into an osteocyte." 

Cartilage canals are formed in existing hyaline cartilage.  To form them you need MT1-MMP(also known as MMP-14) and TRAP.  What exactly is a cartilage canal? 

The role of cartilage canals in endochondral and perichondral bone formation: are there similarities between these two processes? 

"We investigated the development of cartilage canals to clarify their function in the process of bone formation. Cartilage canals are tubes containing vessels that are found in the hyaline cartilage prior to the formation of a secondary ossification centre (SOC). Their exact role is still controversial and it is unclear whether they contribute to endochondral bone formation when an SOC appears. We examined the cartilage canals of the chicken femur in different developmental stages (E20, D2, 5, 7, 8, 10 and 13). To obtain a detailed picture of the cellular and molecular events within and around the canals the femur was investigated by means of three-dimensional reconstruction, light microscopy, electron microscopy, histochemistry and immunohistochemistry [vascular endothelial growth factor (VEGF), type I and II collagen]. An SOC was visible for the first time on the last embryonic day (E20). Cartilage canals were an extension of the vascularized perichondrium[the early form of periosteum] and its mesenchymal stem cell layers into the hyaline cartilage. The canals formed a complex network within the epiphysis and some of them penetrated into the SOC were they ended blind. The growth of the canals into the SOC was promoted by VEGF. As the development progressed the SOC increased in size and adjacent canals were incorporated into it. The canals contained chondroclasts, which opened the lacunae of hypertrophic chondrocytes, and this was followed by invasion of mesenchymal cells into the empty lacunae and formation of an osteoid layer. In older stages this layer mineralized and increased in thickness by addition of further cells. Outside the SOC cartilage canals are surrounded by osteoid, which is formed by the process of perichondral bone formation.  Cartilage canals contribute to both perichondral and endochondral bone formation and osteoblasts have the same origin in both processes." 

So chondrocyte canals actually increased the size of the Secondary Ossification Centers.  VEGF during early development increased height.  Cartilage canals are also formed from periosteum further emphasizing it's importance in height growth.  Therefore, maximizing things like VEGF and MMP-9 during early development may help with height growth by increasing the number of cartilage canals. 

Expression of matrix metalloproteinases during vascularization and ossification of normal and impaired avian growth plate. 

"Enzymes of the matrix metalloproteinase (MMP) family regulate angiogenesis and are involved in the endochondral ossification process. Tibial dyschondroplasia (TD) and rickets are 2 disorders associated with impairments in this process, mainly in the vascularization of the avian growth plate. We induced TD and rickets and studied the expression patterns of 4 members of the MMP family known to be important for endochondral ossification, MMP-2, 3, 9, and 13, in normal and impaired avian growth plates. The expression of MMP-3, 9, and 13 was reduced in the lesions and lined up parallel to the expulsion of blood vessels, which was extended up to the border of the lesion, but did not penetrate into it. Matrix metallopro-teinase-2 was not expressed in the TD lesion but was overexpressed in the rachitic lesion. We also studied the differentiation stage of the chondrocytes populating the lesions and found that the rachitic lesions were populated with proliferative chondrocytes, whereas the TD lesions were filled with chondrocytes that presented both proliferative and hypertrophic markers. MMP-3, 9, and 13 play a role in the vascularization and ossification processes, whereas MMP-2 is related to chondrocyte differentiation and may be involved in cartilage remodeling in the avian growth plate." 

MMP-3, 9, and 13 play a role in the vascularization process and thus in turn the process of forming catilage canals.  But they also play a role in the possibly catabolic process of ossification... 

Akt signaling regulates actin organization via modulation of MMP-2 activity during chondrogenesis of chick wing limb bud mesenchymal cells. 

"Endochondral ossification is initiated by the differentiation of mesenchymal precursor cells to chondrocytes{Note this can occur after fusion}. This process is characterized by a strong interdependence of cell shape and cytoskeletal organization accompanying the onset of chondrogenic gene expression. The activation of matrix metalloproteinase (MMP)-2 [may] be involved in the reorganization of the actin cytoskeleton and that this would require an Akt-dependent signaling pathway in chick wing bud mesenchymal cells. The pharmacological inhibition of Akt signaling resulted in decreased glycosaminoglycan synthesis and reduced the level of active MMP-2, leading to suppressed cortical actin organization which is characteristic of differentiated chondrocytes. The exposure of cells to bafilomycin A1 reversed these chondro-inhibitory effects induced by inhibition of Akt signaling. Akt signaling is involved in the activation of MMP-2 and that this Akt-induced activation of MMP-2 is responsible for reorganization of the actin cytoskeleton into a cortical pattern with parallel rounding of chondrogenic competent cells." 

MMP-2 is crucial for organizing the growth plate by actin based communication.  MMP-2, 3, 9, 13, 14 seem to be important for endochondral ossification.  We already know that Zinc is important.Spring Valley - Zinc 50 mg, 200 Ct. 

Vascular endothelial growth factor (VEGF) induces matrix metalloproteinase expression in immortalized chondrocytes. 

"VEGF (vascular endothelial growth factor), an important angiogenesis factor, appears also to be involved in inflammatory processes. VEGF and its receptors (VEGFR) are expressed on osteoarthritic, but not on normal adult, chondrocytes. The effects of VEGF were studied on immortalized human chondrocytes. Activated matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, tissue inhibitor of metalloproteinases (TIMP)-1, TIMP-2, interleukin (IL)-1beta, IL-6, and tumour necrosis factor-alpha (TNF-alpha) were measured in culture supernatants by enzyme-linked immunosorbent assays, nitric oxide with the Griess reagent, and cell proliferation by [3H]thymidine incorporation. VEGFR-2 mRNA was quantified by real-time reverse transcription-polymerase chain reaction and the protein was identified by immuno-gold electron microscopy. Intracellular signal transduction effects were determined by western blots and electrophoretic mobility shift assays. The chondrocyte cell lines C28/I2, C20/A4, and T/C28a2/a4 expressed functionally active VEGFR-2. VEGF stimulation induced receptor phosphorylation, activation of the mitogen-activated protein kinases ERK 1/2, and long-lasting activation of the transcription factor AP-1 (activator protein-1). VEGF increased secreted MMP-1, MMP-3, and especially MMP-13, which could be effectively reduced by an inhibitor of VEGFR-2 kinase activity. VEGF diminished the expression of TIMP-1 and especially TIMP-2. Under hypoxic conditions, as occur in cartilage, the reduction in TIMP levels was even greater. VEGF induced IL-1beta, IL-6, TNF-alpha, and nitric oxide expression to a small extent and stimulated the proliferation of immortalized chondrocytes. VEGF is an autocrine stimulator of immortalized chondrocytes that mediates mainly destructive processes in osteoarthritis." 

VEGF and MMPs both increase growth by stimulating chondrocyte proliferation even though they increase ossification.  Neither MMP and VEGF inhibition is probably what is causing the 30% height gain in F-spondin inhibition and in fact MMP and VEGF stimulators may increase height growth by increasing chondrocyte proliferation and encouraging the formation of cartilage canals.

Here's something on MMP-3 which is heavily upregulated by LSJL:

Matrix metalloproteinase-3 in articular cartilage is upregulated by joint immobilization and suppressed by passive joint motion.

"Both underloading and overloading of joints can lead to articular cartilage degradation, a process mediated in part by matrix metalloproteinases (MMPs). Here we examine the effects of reduced loading of rat hindlimbs on articular cartilage expression of MMP-3, which not only digests matrix components but also activates other proteolytic enzymes. Hindlimb immobilization resulted in elevated MMP-3 mRNA expression at 6h that was sustained throughout the 21day immobilization period{LSJL does not involve immobilazation, maybe immbolization increases hydrostatic pressure.  Joint movement may help relieve pressure and lack of movement may cause pressure buildup thus LSJL and immobilization may both upregulate MMP-3 by a hydrostatic pressure based mechanism}. MMP-3 upregulation was higher in the medial condyle than the lateral, and was greatest in the superficial cartilage zone, followed by middle and deep zones. These areas also showed decreases in safranin O staining, consistent with reduced cartilage proteoglycan content, as early as 7days after immobilization. One hour of daily moderate mechanical loading, applied as passive joint motion, reduced the MMP-3 and ADAMTS-5 increases that resulted from immobilization, and also prevented changes in safranin O staining. Intra-articular injections of an MMP-3 inhibitor, N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid (NNGH), dampened the catabolic effects of a 7day immobilization period, indicating a likely requirement for MMP-3 in the regulation of proteoglycan levels through ADAMTS-5."

Note though that LSJL involves intermittent loading whereas this, if the immbolization increases hydrostatic pressure hypothesis is correct, is sustained loading thus any damages inferred from immobilization cannot be derived to LSJL due to the fact that with LSJL you get a break from the pressure.

"In cartilage, MMP-3 not only digests many cartilage ECM components, but can also activate the pro-forms of several MMPs and also contribute to the activation of aggrecanase II (ADAMTS-5)"

"we immobilized the right hind limb of rats, which fixed the knee in full flexion"<-this fully flexed state may have increased hydrostatic pressure.

"A significant increase of MMP-3 mRNA expression [occurred] 6 hours after immobilization"<-LSJL is a lot shorter than 6 hours.

MMP-3 though is still the gene that experiences the most substantial upregulation in LSJL.  However, although MMP-3 is catabolic it may be involved in a more remodeling type of adaptation rather than a destructive force.

Age-related changes in the expression of gelatinase and tissue inhibitor of metalloproteinase genes in mandibular condylar, growth plate, and articular cartilage in rats.

"Mandibular condylar cartilage acts as both articular and growth plate cartilage during growth, and then becomes articular cartilage after growth is complete. Cartilaginous extracellular matrix is remodeled continuously via a combination of production, degradation by matrix metalloproteinases (MMPs), and inhibition of MMP activity by tissue inhibitors of metalloproteinases (TIMPs). This study attempted to clarify the age-related changes in the mRNA expression patterns of MMP-2, MMP-9, TIMP-1, TIMP-2, and TIMP-3 in mandibular condylar cartilage in comparison to tibial growth plate and articular cartilage using an in situ hybridization method in growing and adult rats. MMP-2 and MMP-9 were expressed in a wide range of condylar cartilage cells during growth, and their expression domains became limited to mature chondrocytes in adults. The patterns of TIMP-1 and TIMP-2 expression were similar to those of MMP-2 and MMP-9 during growth, and were maintained until adulthood. TIMP-3 was localized to hypertrophic chondrocytes throughout the growth stage. TIMP-1{up, heavily as three different isoforms} and TIMP-2 were general inhibitors of MMP-2{up} and MMP-9 in condylar cartilage, while TIMP-3 regulates the collagenolytic degradation of the hypertrophic cartilage matrix."

MMPs are secreted and activated by Zinc ions.

"MMP-1, -8, and -13 are the collagenases that degrade native collagen fibers. MMP-8 and -13 digest type II collagen molecules with relatively high specificity. MMP-2 degrades type IV collagen fibers and denatured collagens, while MMP-9 digests denatured collagens, type IV collagen, and aggrecan"

"the mandibular condylar cartilage consisted of five cell layers with an erosion zone at the ossification front. The five cell layers were: a fibrous layer with fibroblasts embedded in the fibrous connective tissue; a proliferative cell layer with undifferentiated and proliferative polygonal-shaped cells; a transitional cell layer with flattened cells, without lipid drops in the cytosol; a mature cell layer with ovoid shaped chondrocytes, which were differentiated, and had cell polarity; and a hypertrophic cell layer with enlarged cells with disorganized cytosolic structures"

"When the activity of MMP-13 was inhibited indirectly by the application of fibroblast growth factor 2 (FGF2), the thickness of the cartilaginous cell layers increased"

"MMP-2, MMP-9, TIMP-1, and TIMP-2 interact as general proteolytic enzymes and their inhibitors in cartilage metabolism, while MMP-13 and TIMP-3 may specifically contribute to endochondral bone growth."

MMP driven endochondral fracture union proceeds independently of osteoclast activity.

"We examined a number of anti-resorptive treatments to either block osteoclast activity, including the potent bisphosphonates zoledronic acid (ZA) and clodronate (CLOD), which work via differing mechanisms, or antagonize osteoclastogenesis with recombinant OPG (HuOPG-Fc), comparing these directly to an inhibitor of matrix metalloproteinase (MMP) activity (MMI270). Endochondral ossification to union occurred normally in all anti-resorptive groups. In contrast, MMP inhibition greatly impaired endochondral union, significantly delaying cartilage callus removal. MMP inhibition also produced smaller, denser hard calluses. Hard callus remodeling was, as expected, delayed with ZA, CLOD and OPG treatment at 4 and 6 weeks, resulting in larger more mineralized calluses at 6 weeks. As a result of reduced hard callus turnover, bone formation was reduced with anti-resorptive agents at these time points. These results confirm that the achievement of endochondral fracture union occurs independently of osteoclast activity. Alternatively, MMP secretion by invading cells is obligatory to endochondral union."

"the temporal change in MMP-13 expression during fracture repair coincides with the replacement of cartilage by bone.  MMP-9 (gelatinase-9) removes denatured collagen II fragments along with collagens IV, IV and XI."