Explain the postulated mechanisms behind a protective role of Mg in vascular calcification and critically appraise the level of evidence.
19 Comments
Marwa Alm
Mg prevents VSMC ossification by
primarily directly down regulating pro-calcification pathways (down regulating genes and proteins involved in the transcription of osteogenic genes)
increase abundance of calcification inhibitors (through restoring activity of CaSR- important for matrix Gla proteins (MGP) synthesis-).
The effect of Mg may be intracellular (Mg influx through TRPM7, Which remains uncertain, as other results demonstrated that Mg prevents bovine VSMC calcification even when TRPM7 was blocked by Mg channel inhibitor 2-APB), or may depend on extracellularinhibition of CPP maturation preceding osteogenic transdifferentiation, keeping VSMC in their contractile phenotype.
The experimental setups are often insufficient to distinguish intracellular from extracellular modes of action, both mechanisms are not mutually exclusive.
Mg2 þinhibits the transition from CPP1 towards CPP2 inhibitng VSMC calcification. Increased Mg2þentry facilitated by TRPM7 and possibly angiotensin receptor 2 (AT-2) may directly interfere with Pi-mediated VSMC osteoblast-like transdifferentiation. ,Mg2þmay restore CaSR activity and MGPc-carboxylation or protein expression.
Its animal model studies and therefore need randomised control trial to deduce conclusion
High mg level Prevent conversion CPP1 (circle shape) to CPP2 ( crystals )
Interestingly, a recent study reported that increasing dialysate Mg2þ for 28 days resulted in a 21% re- duction of CPP1 and a 68% reduction of CPP2 [85]. These results suggest that Mg2þ not only inhibits the formation of
High phosphorus concentrations and the absence of inhibitors like Fetuin-A leads to the formation of CCP1, which transforms to CCP2. CCP2 is thought to induce VSMCs and stimulate the procalcification genes like RUNX2i ALPL, and osterix.. The result is VSCM differentiation and calcification with loss of vascular smooth muscle contractility. , calcified VSCMs amplify the calcification process by shedding Ca-loaded exosomes. They are engulğhed by neighbour VSCM. Magnesium is thought to interfere with this process by inhibiting the transition from CPP1 to CPP2.
These are in vitro and animal studies but applying them to humans may be more complex.
the leading hypothesis has been that Mg prevents vascular calcification primarily by downregulating pathways involved in the transcription of osteogenic genes.
A large number of studies have reported that Mg directly downregulates pro-calcification genes and proteins, among others RUNX2, BMP2 and OSX. Other studies report that Mg prevents VSMC calcification through restoring activity of the Ca2þ-sensing receptor (CaSR), important for matrix Gla protein (MGP) synthesis. As such, Mg2þ may increase the abundance of calcification inhibitors. Although these results are often interpreted as direct effects of Mg2þ on gene transcription, it is important to note that the experimental setups are often insufficient to distinguish intracellular from extracellular modes of action. In general, these experiments rely on increasing Pi concentrations in the experimental culture media, which will result in CPP formation in the culture medium. Consequently the effects of Mg2þ may be intracellular, or may depend on extracellular inhibition of CPP maturation, preceding osteogenic transdifferentation. In these setups, measurements of osteoblast-like gene or protein expression is therefore insufficient to draw firm conclusions on the mechanisms involved.
Several studies provide more advanced approaches that support an intracellular role of Mg2þ in the prevention of VSMC transdifferentiation. Knock-down or inhibition of the divalent cation channel transient receptor potential melastatin 7 (TRPM7) impaired the preventive effect of Mg2þ on VSMC calcification . Since TRPM7 is the major Mg2þ channel in VSMCs, these findings demonstrate that inhibition of osteogenic transcription depends on intracellular Mg2þ levels.
Several studies indicate that TRPM7 is required for the anticalcifying effects ofMg2þ in VSMCs. Importantly, in most studies it was not accurately assessed whether Mg2þ entry had been modified by TRPM7 blocking, as no validated intracellular Mg2þ probes or patch-clamp techniques were used. Other studies may suggest an alternative role for TRPM7 in VSMC calcification. Its activation increased after treating VSMCs with interleukin-18, a cytokine that stimulated VSMC calcification effectively in vitro. Taken together, the impact of intracellular Mg2þ on VSMC gene transcription should be studied in more detail by using validated intracellular Mg2þ probes and more specific methods ofTRPM7 blocking.
Influence of magnesium on calciprotein particle formation
In light of recent data demonstrating that CPP2 are
important drivers of VSMC transdifferentiation and vascular calcification, the model describing a major role for Mg2þ on intracellular pro-calcifying pathways might be incomplete. The formation of CPP2 may precede transdifferentiation ofVSMCs into an osteoblast-like phenotype. Consequently the inhibition of CPP2 maturation by Mg2þ will then result in reduced osteogenic gene expression and maintain VSMCs in their contractile phenotype. The importance of these mechanisms in the context of
CKD-induced vascular calcification was recently tested by our group and by others. Interestingly, Mg2þ did not inhibit initial CPP1 formation from supersaturated concentrations of Ca2þ and Pi but prevented maturation into crystalline CPP2 . Once CPP2 was formed, Mg2þ was incapable of preventing CPP2-induced VSMC mineralization and osteogenic protein expression. Therefore these findings suggest that Mg2þ-dependent inhibition of CPP2 maturation is instrumental in the prevention ofVSMC calcification. Mg2þ not only inhibits the formation ofCPP2 formation, but also CPP1, and translates to patients, lowering the overall CPP burden in CKD. In this context, it is important to realize that intracellular
transcriptional effects of Mg2þ and extracellular Mg2þ effects on CPP maturation are not mutually exclusive. Both mechanisms may contribute to reduced vascular calcification in CKD.
Explain the postulated mechanisms behind a protective role of Mg in vascular calcification and critically appraise the level of evidence.
· Mg prevents vascular calcification primarily by downregulating pathways involved in the transcription of osteogenic genes. A large number of studies have reported that Mg directly downregulates pro-calcification genes and proteins, among others RUNX2, BMP2 and OSX
· Other studies report that Mg prevents VSMC calcification through restoring activity of the Ca2þ-sensing receptor (CaSR), important for matrix Gla protein (MGP) synthesis. Mg may increase the abundance of calcification inhibitors. Although these results are often interpreted as direct effects of Mg on gene transcription
· Mg may increase the abundance of calcification inhibitors.Although these results are often interpreted as direct effects of Mg on gene transcription, it is important to note that the experimental setups are often insufficient to distinguish intracellular from extracellular modes of action. In general, these experiments rely on increasing Pi concentrations in the experimental culture media, which will result in CPP formation in the culture medium. Consequently the effects of Mg may be intracellular, or may depend on extracellular inhibition of CPP maturation, preceding osteogenic transdifferentation
· Mg supplementation is a promising treatment option for vascular calcification. Inhibition of CPP2 maturation may be the key molecular mechanism that explains the prevention of vascular calcification by Mg
· – Several studies indicate that TRPM7 is required for the anticalcifying effects of Mg in VSMCs. hese studies demonstrate the need for intracellular Mg to limit VSMC calcification. Interestingly, other studies have shown that TRPM7and increased Mg influx may mediate the anti-calcifying properties of angiotensin type 2
· In Summary , High Pi concentrations and the absence of circulating inhibitors such as fetuin-A stimulate the formation of CPP1 in the circulation of CKD patients. Subsequently CPP1 transitions into CPP2 due to a lack of circulating crystallization inhibitors – In vitro studies suggest that CPP2 and Pi induce calcification in VSMCs and stimulate expression of pro-calcification genes such as RUNX2, ALP (ALPL) and osterix (SP7).
· Simultaneously, contractility genes such as transgelin (SM22a) diminish.Combined, this cascade results in VSMC transdifferentiation and calcification and loss of VSMC function and contractility. 4 – The resulting calcified VSMCs amplify the calcification process by shedding Caloaded exosomes that are engulfed by neighbouring VSMCs. Mg2þ is proposed to interfere with the calcification process on multiple levels.
·-First, Mg inhibits the transition from CPP1 towards CPP2, preventing VSMC calcification. Second, increased Mg entry facilitated by TRPM7 and possibly angiotensin receptor 2 (AT-2) may directly interfere with Pi-mediated VSMC osteoblast-like transdifferentiation. Third, Mg may restore CaSR activity and MGP c-carboxylation or protein expression.
·- According to level of evedince still undertrial in animal experimental model not reach to the cut point .
Mg is proven to inhibit conversion of CPP1 to CPP2 and thus retard crystallization of Ca and PO4 in soft tissue and vascular smooth muscle cells.
Mg restores activity of CaSR thus restoring synthesis of matrix Gla protein Mg also inhibits the wnt/β-catenin signaling pathway and thus inhibits vascular calcification
It’s possible that studies on animals aren’t a good indicator of how humans would react. Several studies are launched to demonstrate whether Mg supplementation can inhibit the progression of VC in patients with CKD. So far there is no conclusive evidence Mg is involved in VC through a variety of molecular mechanisms.Mg2+ replaces Ca ions in the structure of hydroxyapatite leading to a loss of its crystallinity and inhibiting the maturation of calmodulin particles, thus inhibiting VC. Mg inhibits the wnt/β-catenin signaling pathway to reduce VC
In cellular and rodent models, Mg supplementation prevented high Pi-induced vascular calcification. Mg supplementation to Pi and Ca– enriched culture medium effectively suppressed calcification in human, bovine and rat VSMCs in vitro.
Mg prevented calcification in rat aortic rings treated with high Pi and in a rat model of CKD .
vascular calcification in Klotho knock-out mice was abrogated by a high-Mg diet.
Mg may increase the risk for osteomalacia in CKD by disturbing bone mineralization when used in too high concentrations or during development .
The anti-calcifying properties ofMg are operational at the medial layer of arteries .A substantial body of studies has investigated potential mechanisms underlying these protective properties. Recent insights regarding the impact of Mg on CPP maturation expanded the spectrum of mechanisms that underlie the anti-calcifying properties of Mg.
Influence of magnesium on VSMC transdifferentiation
VSMC transdifferentiation towards an osteoblast-like cell has often been suggested to be the driving event of vascular calcification, manifestation and progression. Consequently the leading hypothesis has been that Mg prevents vascular calcification primarily by downregulating pathways involved in the transcription of osteogenic genes.
A large number of studies have reported that Mg directly downregulates pro-calcification genes and proteins, among others RUNX2, BMP2 and OSX .
Other studies report that Mg prevents VSMC calcification through restoring activity of the Ca2þ-sensing receptor (CaSR), important for matrix Gla protein (MGP) synthesis .
Mg may increase the abundance of calcification inhibitors.
these experiments rely on increasing Pi concentrations in the experimental culture media, which will result in CPP formation in the culture medium.
Consequently the effects of Mg may be intracellular, or may depend on extracellular inhibition of CPP maturation, preceding osteogenic transdifferentation .
In these setups, measurements of osteoblast-like gene or protein expression is therefore insufficient to draw firm conclusions on the mechanisms involved.
Several studies provide more advanced approaches that support an intracellular role of Mg in the prevention of VSMC transdifferentiation. Knock-down or inhibition of the divalent cation channel transient receptor potential melastatin 7 (TRPM7) impaired the preventive effect of Mg on VSMC calcification .
Since TRPM7 is the major Mg channel in VSMCs, these findings demonstrate that inhibition of osteogenic transcription depends on intracellular Mg levels.
Several studies indicate that TRPM7 is required for the anti- calcifying effects of Mg in VSMCs.
In addition, treatment of human VSMCs with a small interfering RNA directed at TRPM7 resulted in calcification despite Mg supplementation.
Combined, these studies demonstrate the need for intracellular Mg to limit VSMC calcification. Interestingly, other studies have shown that TRPM7 and increased Mg influx may mediate the anti-calcifying properties of angiotensin type 2 .
the obligate involvement of TRPM7 remains uncertain, as results from our group demonstrated that Mg actually did prevent bovine VSMC calcification even when TRPM7 was blocked using the channel inhibitor 2-APB .While similar concentrations were used, the use of bovine VSMCs rather than human VSMCs might explain these contradictory findings.
Other studies may suggest an alternative role for TRPM7 in VSMC calcification. Its activation increased after treating VSMCs with interleukin-18, a cytokine that stimulated VSMC calcification effectively in vitro .
Taken together, the impact of intracellular Mg on VSMC gene transcription should be studied in more detail by using validated intracellular Mg probes and more specific methods of TRPM7 blocking.
Influence of magnesium on calciprotein particle formation
The effect of Mg on hydroxyapatite formation was widely studied in the 1970s. Although not in the context of CKD, the interference on Ca–Pi crystallization by Mg has been established in several experimental studies.
First, Mg can substitute Ca in hydroxyapatite formation, favouring the formation of Mg-containing whitlockite instead .
Second, Mg may prevent the formation of hydroxyapatite altogether .
The importance of these mechanisms in the context of CKD-induced vascular calcification was recently tested by our group and by others.
Interestingly, Mg did not inhibit initial CPP1 formation from supersaturated concentrations of Ca and Pi but prevented maturation into crystalline CPP2 .
Once CPP2 was formed, Mg was incapable of preventing CPP2-induced VSMC mineralization and osteogenic protein expression.
Therefore these findings suggest that Mg-dependent inhibition of CPP2 maturation is instrumental in the prevention of VSMC calcification .
These results suggest that Mg not only inhibits the formation of CPP2 formation, but also CPP1, and translates to patients, lowering the overall CPP burden in CKD.
In this context, it is important to realize that intracellular transcriptional effects of Mg and extracellular Mg effects on CPP maturation are not mutually exclusive. Both mechanisms may contribute to reduced vascular calcification in CKD.
The VSMC transdifferentiate into osteoblast like cell which is the driving force for the vascular calcification, manifestation and progression.
The role of the Mg is the downregulation of the pro-calcification genes and protein RUNX2, BMP, osteopontin and OSX.
Studies show that Mg prevent VSMC calcification through the restoration of the activity of CaSR which is important for matrix Gla protein synthesis MGP.
Also Mg can increase the abundance of calcification inhibitors.
The effect of Mg can be intracellular or extracellular inhibitions of CCP formation.
Knock-down the divalent cation channel TRPM7 will impair the preventive effect of Mg on VSMC calcification, so that inhibition of osteogenic transcription depends on intracellular Mg level.
TRPM7 and high Mg levels will increase the calcifying properties of angioteinsin type.
The CPP2 formation occur before VSMC transdifferentiation, Mg inhibit the CPP2 formation,first the Mg will substitute the Calcium in hydroxy apatite formation and lead to Mg containing whitelockite which is less pathogenic, second Mg inhibit hydroxy apatite formation.
In aqueous solution Mg will shield the amorphous CCP1 from transition to crystalline form which will induce VSMC mineralization and osteogenic protein expression, because once the CPP2 formed it will progress regardless the level of Mg.
Recent studies show that increase Mg in the dialysate for 28 days lead to 21%and 68% reduction of CCP 1 and CCP 2 respectively.
Explain the postulated mechanisms behind a protective role of Mg in vascular calcification ( VC) and critically appraise the level of evidence.
-Mg is involved in VC through a variety of molecular mechanisms. – Mg2+ replaces Ca ions in the structure of hydroxyapatite leading to a loss of its crystallinity and inhibiting the maturation of calmodulin particles, thus inhibiting VC. – Mg inhibits the wnt/β-catenin signaling pathway to reduce VC
Several studies are launched to demonstrate whether Mg supplementation can inhibit the progression of VC in patients with CKD.So far there is no conclusive evidenceز
The mechanisms behind a protective role of Mg in vascular calcification
Inhibit smooth muscle transdifferntiation via inhibiton of transcription of osteogenic genes .
And restoration of the activity of CaSR, this is very important for the synsthisis inhibitor of the vascular calcification.
According to level of evedince still undertrial in animal experimental model not reach to cut point and clue about proper level of mg
Magnesium (Mg) has been postulated to have a protective role in vascular calcification by various mechanisms. One mechanism is the downregulation of osteogenic gene transcription, such as RUNX2, BMP2, and OSX, which are responsible for promoting the formation of bone-like structures in the arteries. Mg also inhibits the expression of proteins and enzymes that promote calcification, such as alkaline phosphatase, osteopontin, and matrix Gla protein. Furthermore, Mg is known to restore the activity of the calcium-sensing receptor (CaSR), which is essential for maintaining normal calcium homeostasis and preventing VSMC calcification.
magnesium can fight the 2 main pathway of vascular calcification:
1- Mg affect cpp by 2 mechanisms: i-Mg substitutes ca in hydroxyaoaoatite forming less toxix Mg containing whitlcockite, ii- Mg prevent formation of hydroxyapatite completely
2- Mg affects VSMC transdifferentiation into osteoblast-like cells by downregulating transcription of osteogenic genes and increase the calcification inhibitors
The protective role of magnesium against vascular calcification
Progression of CKD is associated with an increased risk of vascular calcification.
In dialysis patients, the prevalence of vascular calcification is approximately >80%.
Intimal calcification develops at the site of atheromatous plaques and is usually associated with advanced stages of atherosclerosis.
Medial calcification is associated with the metabolic imbalance (mineral and glucose) in the larger arteries and is usually associated with aging, diabetes, and CKD.
Vascular calcification is considered to be irreversible.
Measures to prevent vascular calcification;
Reduce phosphorus level.
Use calcium-free phosphate binders.
VitD3.
ADVANCE trial reported promising effects of a low dose of vitD3in combination with cinacalcet on coronary, aortic, and cardiac valve calcification progression.
Hypomagnesemia is a well-known risk factor for cardiovascular disease, events, and mortality.
Low dietary Mg is associated with increased all-cause mortality, risk of stroke, heart failure, and DM, and a similar association occurs in CKD and dialysis patients.
An observation study showed that a higher magnesium level is associated with a reduced risk of developing vascular calcification.
Serum Mg of <1.14 mmol/l in dialysis patients is associated with a higher risk of mortality.
An experimental study in mice showed Kloth knock-out properties with a high serum Mg level.
The anti-calcific properties of Mg occur at the medial layer of arteries.
The leading hypothesis suggests that Mg prevents vascular calcification by downregulation of the transcription properties of osteogenic genes.
A larger number of studies show that Mg downregulates pro-calcification genes and proteins among others RUNX2, BMP2, and OSX.
Other study reports that Mg prevents VSMC calcification by restoring the activity of the CaSR, important for matrix G1a protein synthesis.
Many reports explain that the higher concentration of magnesium level behind protective role of Mg in vascular calcification by two mechanism 1- Mg prevent osteogenic vascular smooth muscles cell transdifferentiation invitro and in vivo modle 2- other study show Mg prevent calciprotien particles maturation
The protective role of Mg in vascular calcification
In CKD and dialysis, greater serum Mg2+ concentrations are related to decreased cardiovascular morbidity and mortality. Several experimental models have studied the possible preventative benefit of Mg2+.
Mg2+ inhibited high Pi-induced vascular calcification in cells and rodents. Mg2+ addition to Pi and Ca2+-enriched culture media inhibited calcification in human, bovine, and rat VSMCs.
Mg2+ inhibited calcification in high-Pi-treated rat aortic rings and CKD rats.
High-Mg2 diets prevented vascular calcification in Klotho knock-out mice. Mg2+ in high amounts or during development may cause osteomalacia in CKD by disrupting bone mineralization. While longer-term studies are needed, a 28-day increase in dialysate Mg2+ concentration (1.0 vs 0.5 mmol/L) did not modify bone turnover indicators.
Mg2+ inhibits vascular calcification by downregulating osteogenic gene transcription. Mg2+ directly downregulates pro-calcification genes and proteins such as RUNX2, BMP2, and OSX.
Vascular calcification therapy with Mg2+ is promising. Mg2 may prevent vascular calcification by inhibiting CPP2 maturation and improving VSMC gene expression.
The level of evidence:It’s possible that studies on animals aren’t a good indicator of how humans would react.
-short-term follow-up.
Explain the postulated mechanisms behind a protective role of Mg in vascular calcification. critically appraise the level of evidence.
Down regulation of transcription of osteogenic genes e.g., RUNX2, BMP2 & OSX
Restoration of the activity of CaSR, this is very important for the synthesis of matrix Gla protein (MGP) a potent inhibitor of the vascular calcification
Critically appraise the level of evidence.
Experimental set up may be be adequate to differentiate between intracellular & extracellular mode of actions and therefore, it is insufficient to draw a very strong conclusions about the exact mechanism involved.
Mg prevents VSMC ossification by
The effect of Mg may be intracellular (Mg influx through TRPM7, Which remains uncertain, as other results demonstrated that Mg prevents bovine VSMC calcification even when TRPM7 was blocked by Mg channel inhibitor 2-APB), or may depend on extracellularinhibition of CPP maturation preceding osteogenic transdifferentiation, keeping VSMC in their contractile phenotype.
The experimental setups are often insufficient to distinguish intracellular from extracellular modes of action, both mechanisms are not mutually exclusive.
Mg2 þinhibits the transition from CPP1 towards CPP2 inhibitng VSMC calcification. Increased Mg2þentry facilitated by TRPM7 and possibly angiotensin receptor 2 (AT-2) may directly interfere with Pi-mediated VSMC osteoblast-like transdifferentiation. ,Mg2þmay restore CaSR activity and MGPc-carboxylation or protein expression.
Its animal model studies and therefore need randomised control trial to deduce conclusion
High mg level Prevent conversion CPP1 (circle shape) to CPP2 ( crystals )
Interestingly, a recent study reported that increasing dialysate Mg2þ for 28 days resulted in a 21% re- duction of CPP1 and a 68% reduction of CPP2 [85]. These results suggest that Mg2þ not only inhibits the formation of
High phosphorus concentrations and the absence of inhibitors like Fetuin-A leads to the formation of CCP1, which transforms to CCP2. CCP2 is thought to induce VSMCs and stimulate the procalcification genes like RUNX2i ALPL, and osterix.. The result is VSCM differentiation and calcification with loss of vascular smooth muscle contractility. , calcified VSCMs amplify the calcification process by shedding Ca-loaded exosomes. They are engulğhed by neighbour VSCM. Magnesium is thought to interfere with this process by inhibiting the transition from CPP1 to CPP2.
These are in vitro and animal studies but applying them to humans may be more complex.
the leading hypothesis has been that Mg prevents vascular calcification primarily by downregulating pathways involved in the transcription of osteogenic genes.
A large number of studies have reported that Mg directly downregulates pro-calcification genes and proteins, among others RUNX2, BMP2 and OSX. Other studies report that Mg prevents VSMC calcification through restoring activity of the Ca2þ-sensing receptor (CaSR), important for matrix Gla protein (MGP) synthesis. As such, Mg2þ may increase the abundance of calcification inhibitors. Although these results are often interpreted as direct effects of Mg2þ on gene transcription, it is important to note that the experimental setups are often insufficient to distinguish intracellular from extracellular modes of action. In general, these experiments rely on increasing Pi concentrations in the experimental culture media, which will result in CPP formation in the culture medium. Consequently the effects of Mg2þ may be intracellular, or may depend on extracellular inhibition of CPP maturation, preceding osteogenic transdifferentation. In these setups, measurements of osteoblast-like gene or protein expression is therefore insufficient to draw firm conclusions on the mechanisms involved.
Several studies provide more advanced approaches that support an intracellular role of Mg2þ in the prevention of VSMC transdifferentiation. Knock-down or inhibition of the divalent cation channel transient receptor potential melastatin 7 (TRPM7) impaired the preventive effect of Mg2þ on VSMC calcification . Since TRPM7 is the major Mg2þ channel in VSMCs, these findings demonstrate that inhibition of osteogenic transcription depends on intracellular Mg2þ levels.
Several studies indicate that TRPM7 is required for the anticalcifying effects ofMg2þ in VSMCs. Importantly, in most studies it was not accurately assessed whether Mg2þ entry had been modified by TRPM7 blocking, as no validated intracellular Mg2þ probes or patch-clamp techniques were used. Other studies may suggest an alternative role for TRPM7 in VSMC calcification. Its activation increased after treating VSMCs with interleukin-18, a cytokine that stimulated VSMC calcification effectively in vitro. Taken together, the impact of intracellular Mg2þ on VSMC gene transcription should be studied in more detail by using validated intracellular Mg2þ probes and more specific methods ofTRPM7 blocking.
Influence of magnesium on calciprotein particle formation
In light of recent data demonstrating that CPP2 are
important drivers of VSMC transdifferentiation and vascular calcification, the model describing a major role for Mg2þ on intracellular pro-calcifying pathways might be incomplete. The formation of CPP2 may precede transdifferentiation ofVSMCs into an osteoblast-like phenotype. Consequently the inhibition of CPP2 maturation by Mg2þ will then result in reduced osteogenic gene expression and maintain VSMCs in their contractile phenotype. The importance of these mechanisms in the context of
CKD-induced vascular calcification was recently tested by our group and by others. Interestingly, Mg2þ did not inhibit initial CPP1 formation from supersaturated concentrations of Ca2þ and Pi but prevented maturation into crystalline CPP2 . Once CPP2 was formed, Mg2þ was incapable of preventing CPP2-induced VSMC mineralization and osteogenic protein expression. Therefore these findings suggest that Mg2þ-dependent inhibition of CPP2 maturation is instrumental in the prevention ofVSMC calcification. Mg2þ not only inhibits the formation ofCPP2 formation, but also CPP1, and translates to patients, lowering the overall CPP burden in CKD. In this context, it is important to realize that intracellular
transcriptional effects of Mg2þ and extracellular Mg2þ effects on CPP maturation are not mutually exclusive. Both mechanisms may contribute to reduced vascular calcification in CKD.
Explain the postulated mechanisms behind a protective role of Mg in vascular calcification and critically appraise the level of evidence.
· Mg prevents vascular calcification primarily by downregulating pathways involved in the transcription of osteogenic genes. A large number of studies have reported that Mg directly downregulates pro-calcification genes and proteins, among others RUNX2, BMP2 and OSX
· Other studies report that Mg prevents VSMC calcification through restoring activity of the Ca2þ-sensing receptor (CaSR), important for matrix Gla protein (MGP) synthesis. Mg may increase the abundance of calcification inhibitors. Although these results are often interpreted as direct effects of Mg on gene transcription
· Mg may increase the abundance of calcification inhibitors.Although these results are often interpreted as direct effects of Mg on gene transcription, it is important to note that the experimental setups are often insufficient to distinguish intracellular from extracellular modes of action. In general, these experiments rely on increasing Pi concentrations in the experimental culture media, which will result in CPP formation in the culture medium. Consequently the effects of Mg may be intracellular, or may depend on extracellular inhibition of CPP maturation, preceding osteogenic transdifferentation
· Mg supplementation is a promising treatment option for vascular calcification. Inhibition of CPP2 maturation may be the key molecular mechanism that explains the prevention of vascular calcification by Mg
· – Several studies indicate that TRPM7 is required for the anticalcifying effects of Mg in VSMCs. hese studies demonstrate the need for intracellular Mg to limit VSMC calcification. Interestingly, other studies have shown that TRPM7and increased Mg influx may mediate the anti-calcifying properties of angiotensin type 2
· In Summary , High Pi concentrations and the absence of circulating inhibitors such as fetuin-A stimulate the formation of CPP1 in the circulation of CKD patients. Subsequently CPP1 transitions into CPP2 due to a lack of circulating crystallization inhibitors – In vitro studies suggest that CPP2 and Pi induce calcification in VSMCs and stimulate expression of pro-calcification genes such as RUNX2, ALP (ALPL) and osterix (SP7).
· Simultaneously, contractility genes such as transgelin (SM22a) diminish.Combined, this cascade results in VSMC transdifferentiation and calcification and loss of VSMC function and contractility. 4 – The resulting calcified VSMCs amplify the calcification process by shedding Caloaded exosomes that are engulfed by neighbouring VSMCs. Mg2þ is proposed to interfere with the calcification process on multiple levels.
·-First, Mg inhibits the transition from CPP1 towards CPP2, preventing VSMC calcification. Second, increased Mg entry facilitated by TRPM7 and possibly angiotensin receptor 2 (AT-2) may directly interfere with Pi-mediated VSMC osteoblast-like transdifferentiation. Third, Mg may restore CaSR activity and MGP c-carboxylation or protein expression.
·- According to level of evedince still undertrial in animal experimental model not reach to the cut point .
Mg is proven to inhibit conversion of CPP1 to CPP2 and thus retard crystallization of Ca and PO4 in soft tissue and vascular smooth muscle cells.
Mg restores activity of CaSR thus restoring synthesis of matrix Gla protein
Mg also inhibits the wnt/β-catenin signaling pathway and thus inhibits vascular calcification
It’s possible that studies on animals aren’t a good indicator of how humans would react. Several studies are launched to demonstrate whether Mg supplementation can inhibit the progression of VC in patients with CKD. So far there is no conclusive evidence
Mg is involved in VC through a variety of molecular mechanisms. Mg2+ replaces Ca ions in the structure of hydroxyapatite leading to a loss of its crystallinity and inhibiting the maturation of calmodulin particles, thus inhibiting VC.
Mg inhibits the wnt/β-catenin signaling pathway to reduce VC
In cellular and rodent models, Mg supplementation prevented high Pi-induced vascular calcification. Mg supplementation to Pi and Ca– enriched culture medium effectively suppressed calcification in human, bovine and rat VSMCs in vitro.
Mg prevented calcification in rat aortic rings treated with high Pi and in a rat model of CKD .
vascular calcification in Klotho knock-out mice was abrogated by a high-Mg diet.
Mg may increase the risk for osteomalacia in CKD by disturbing bone mineralization when used in too high concentrations or during development .
The anti-calcifying properties ofMg are operational at the medial layer of arteries .A substantial body of studies has investigated potential mechanisms underlying these protective properties. Recent insights regarding the impact of Mg on CPP maturation expanded the spectrum of mechanisms that underlie the anti-calcifying properties of Mg.
Influence of magnesium on VSMC transdifferentiation
VSMC transdifferentiation towards an osteoblast-like cell has often been suggested to be the driving event of vascular calcification, manifestation and progression. Consequently the leading hypothesis has been that Mg prevents vascular calcification primarily by downregulating pathways involved in the transcription of osteogenic genes.
A large number of studies have reported that Mg directly downregulates pro-calcification genes and proteins, among others RUNX2, BMP2 and OSX .
Other studies report that Mg prevents VSMC calcification through restoring activity of the Ca2þ-sensing receptor (CaSR), important for matrix Gla protein (MGP) synthesis .
Mg may increase the abundance of calcification inhibitors.
these experiments rely on increasing Pi concentrations in the experimental culture media, which will result in CPP formation in the culture medium.
Consequently the effects of Mg may be intracellular, or may depend on extracellular inhibition of CPP maturation, preceding osteogenic transdifferentation .
In these setups, measurements of osteoblast-like gene or protein expression is therefore insufficient to draw firm conclusions on the mechanisms involved.
Several studies provide more advanced approaches that support an intracellular role of Mg in the prevention of VSMC transdifferentiation. Knock-down or inhibition of the divalent cation channel transient receptor potential melastatin 7 (TRPM7) impaired the preventive effect of Mg on VSMC calcification .
Since TRPM7 is the major Mg channel in VSMCs, these findings demonstrate that inhibition of osteogenic transcription depends on intracellular Mg levels.
Several studies indicate that TRPM7 is required for the anti- calcifying effects of Mg in VSMCs.
In addition, treatment of human VSMCs with a small interfering RNA directed at TRPM7 resulted in calcification despite Mg supplementation.
Combined, these studies demonstrate the need for intracellular Mg to limit VSMC calcification. Interestingly, other studies have shown that TRPM7 and increased Mg influx may mediate the anti-calcifying properties of angiotensin type 2 .
the obligate involvement of TRPM7 remains uncertain, as results from our group demonstrated that Mg actually did prevent bovine VSMC calcification even when TRPM7 was blocked using the channel inhibitor 2-APB .While similar concentrations were used, the use of bovine VSMCs rather than human VSMCs might explain these contradictory findings.
Other studies may suggest an alternative role for TRPM7 in VSMC calcification. Its activation increased after treating VSMCs with interleukin-18, a cytokine that stimulated VSMC calcification effectively in vitro .
Taken together, the impact of intracellular Mg on VSMC gene transcription should be studied in more detail by using validated intracellular Mg probes and more specific methods of TRPM7 blocking.
Influence of magnesium on calciprotein particle formation
The effect of Mg on hydroxyapatite formation was widely studied in the 1970s. Although not in the context of CKD, the interference on Ca–Pi crystallization by Mg has been established in several experimental studies.
First, Mg can substitute Ca in hydroxyapatite formation, favouring the formation of Mg-containing whitlockite instead .
Second, Mg may prevent the formation of hydroxyapatite altogether .
The importance of these mechanisms in the context of CKD-induced vascular calcification was recently tested by our group and by others.
Interestingly, Mg did not inhibit initial CPP1 formation from supersaturated concentrations of Ca and Pi but prevented maturation into crystalline CPP2 .
Once CPP2 was formed, Mg was incapable of preventing CPP2-induced VSMC mineralization and osteogenic protein expression.
Therefore these findings suggest that Mg-dependent inhibition of CPP2 maturation is instrumental in the prevention of VSMC calcification .
These results suggest that Mg not only inhibits the formation of CPP2 formation, but also CPP1, and translates to patients, lowering the overall CPP burden in CKD.
In this context, it is important to realize that intracellular transcriptional effects of Mg and extracellular Mg effects on CPP maturation are not mutually exclusive. Both mechanisms may contribute to reduced vascular calcification in CKD.
The VSMC transdifferentiate into osteoblast like cell which is the driving force for the vascular calcification, manifestation and progression.
The role of the Mg is the downregulation of the pro-calcification genes and protein RUNX2, BMP, osteopontin and OSX.
Studies show that Mg prevent VSMC calcification through the restoration of the activity of CaSR which is important for matrix Gla protein synthesis MGP.
Also Mg can increase the abundance of calcification inhibitors.
The effect of Mg can be intracellular or extracellular inhibitions of CCP formation.
Knock-down the divalent cation channel TRPM7 will impair the preventive effect of Mg on VSMC calcification, so that inhibition of osteogenic transcription depends on intracellular Mg level.
TRPM7 and high Mg levels will increase the calcifying properties of angioteinsin type.
The CPP2 formation occur before VSMC transdifferentiation, Mg inhibit the CPP2 formation,first the Mg will substitute the Calcium in hydroxy apatite formation and lead to Mg containing whitelockite which is less pathogenic, second Mg inhibit hydroxy apatite formation.
In aqueous solution Mg will shield the amorphous CCP1 from transition to crystalline form which will induce VSMC mineralization and osteogenic protein expression, because once the CPP2 formed it will progress regardless the level of Mg.
Recent studies show that increase Mg in the dialysate for 28 days lead to 21%and 68% reduction of CCP 1 and CCP 2 respectively.
Explain the postulated mechanisms behind a protective role of Mg in vascular calcification ( VC) and critically appraise the level of evidence.
-Mg is involved in VC through a variety of molecular mechanisms.
– Mg2+ replaces Ca ions in the structure of hydroxyapatite leading to a loss of its crystallinity and inhibiting the maturation of calmodulin particles, thus inhibiting VC.
– Mg inhibits the wnt/β-catenin signaling pathway to reduce VC
Several studies are launched to demonstrate whether Mg supplementation can inhibit the progression of VC in patients with CKD.So far there is no conclusive evidenceز
1- Mg inhibits the transition from CPP1 towards CPP2, preventing VSMC calcification.
2- increased Mg entry facilitated by TRPM7 and possibly angiotensin receptor 2 (AT-2) may directly interfere with Pi-mediated VSMC osteoblast-like transdifferentiation.
3- Mg may restore CaSR activity and MGP c-carboxylation or protein expression.
The mechanisms behind a protective role of Mg in vascular calcification
Inhibit smooth muscle transdifferntiation via inhibiton of transcription of osteogenic genes .
And restoration of the activity of CaSR, this is very important for the synsthisis inhibitor of the vascular calcification.
According to level of evedince still undertrial in animal experimental model not reach to cut point and clue about proper level of mg
Magnesium (Mg) has been postulated to have a protective role in vascular calcification by various mechanisms. One mechanism is the downregulation of osteogenic gene transcription, such as RUNX2, BMP2, and OSX, which are responsible for promoting the formation of bone-like structures in the arteries. Mg also inhibits the expression of proteins and enzymes that promote calcification, such as alkaline phosphatase, osteopontin, and matrix Gla protein. Furthermore, Mg is known to restore the activity of the calcium-sensing receptor (CaSR), which is essential for maintaining normal calcium homeostasis and preventing VSMC calcification.
The level of evidence is mixed
magnesium can fight the 2 main pathway of vascular calcification:
1- Mg affect cpp by 2 mechanisms: i-Mg substitutes ca in hydroxyaoaoatite forming less toxix Mg containing whitlcockite, ii- Mg prevent formation of hydroxyapatite completely
2- Mg affects VSMC transdifferentiation into osteoblast-like cells by downregulating transcription of osteogenic genes and increase the calcification inhibitors
The protective role of magnesium against vascular calcification
Many reports explain that the higher concentration of magnesium level behind protective role of Mg in vascular calcification
by two mechanism
1- Mg prevent osteogenic vascular smooth muscles cell transdifferentiation
invitro and in vivo modle
2- other study show Mg prevent calciprotien particles maturation
The protective role of Mg in vascular calcification
In CKD and dialysis, greater serum Mg2+ concentrations are related to decreased cardiovascular morbidity and mortality. Several experimental models have studied the possible preventative benefit of Mg2+.
Mg2+ inhibited high Pi-induced vascular calcification in cells and rodents. Mg2+ addition to Pi and Ca2+-enriched culture media inhibited calcification in human, bovine, and rat VSMCs.
Mg2+ inhibited calcification in high-Pi-treated rat aortic rings and CKD rats.
High-Mg2 diets prevented vascular calcification in Klotho knock-out mice. Mg2+ in high amounts or during development may cause osteomalacia in CKD by disrupting bone mineralization. While longer-term studies are needed, a 28-day increase in dialysate Mg2+ concentration (1.0 vs 0.5 mmol/L) did not modify bone turnover indicators.
Mg2+ inhibits vascular calcification by downregulating osteogenic gene transcription. Mg2+ directly downregulates pro-calcification genes and proteins such as RUNX2, BMP2, and OSX.
Vascular calcification therapy with Mg2+ is promising. Mg2 may prevent vascular calcification by inhibiting CPP2 maturation and improving VSMC gene expression.
The level of evidence:It’s possible that studies on animals aren’t a good indicator of how humans would react.
-short-term follow-up.
Explain the postulated mechanisms behind a protective role of Mg in vascular calcification. critically appraise the level of evidence.
Critically appraise the level of evidence.