Jonathan C. Vacek

MMP-2/TIMP-2/TIMP-4 versus MMP-9/TIMP-3 in transition from compensatory hypertrophy and angiogenesis to decompensatory heart failure*

Although matrix metalloproteinase (MMPs) and tissue inhibitor of metalloproteinase (TIMPs) play a vital role in tumour angiogenesis and TIMP-3 caused apoptosis, their role in cardiac angiogenesis is unknown. Interestingly, a disruption of co-ordinated cardiac hypertrophy and angiogenesis contributed to the transition to heart failure, however, the proteolytic and anti-angiogenic mechanisms of transition from compensatory hypertrophy to decompensatory heart failure were unclear. We hypothesized that after an aortic stenosis MMP-2 released angiogenic factors during compensatory hypertrophy and MMP-9/TIMP-3 released anti-angiogenic factors causing decompensatory heart failure. To verify this hypothesis, wild type (WT) mice were studied 3 and 8 weeks after aortic stenosis, created by banding the ascending aorta in WT and MMP-9-/- (MMP-9KO) mice. Cardiac function (echo, PV loops) was decreased at 8 weeks after stenosis. The levels of MMP-2 (western blot) increased at 3 weeks and returned to control level at 8 weeks, MMP-9 increased only at 8 weeks. TIMP-2 and -4 decreased at 3 and even more at 8 weeks. The angiogenic VEGF increased at 3 weeks and decreased at 8 weeks, the anti-angiogenic endostatin and angiostatin increased only at 8 weeks. CD-31 positive endothelial cells were more intensely labelled at 3 weeks than in sham operated or in 8 weeks banded mice. Vascularization, as estimated by x-ray angiography, was increased at 3 weeks and decreased at 8 weeks post-banding. Although the vast majority of studies were performed on control WT mice only, interestingly, MMP9-KO mice seemed to have increased vascular density 8 weeks after banding. These results suggested that there was increase in MMP-2, decrease in TIMP-2 and -4, increase in angiogenic factors and vascularization in compensatory hearts. However, in decompensatory hearts there was increase in MMP-9, TIMP-3, endostatin, angiostatin and vascular rarefaction.

Hydrogen sulfide mitigates transition from compensatory hypertrophy to heart failure

We reported previously that although there is disruption of coordinated cardiac hypertrophy and angiogenesis in transition to heart failure, matrix metalloproteinase (MMP)-9 induced antiangiogenic factors play a vital role in this process. Previous studies have shown the cardioprotective role of hydrogen sulfide (H₂S) in various cardiac diseases, but its role during transition from compensatory hypertrophy to heart failure is yet to be unveiled. We hypothesize that H₂S induces MMP-2 activation and inhibits MMP-9 activation, thus promoting angiogenesis, and mitigates transition from compensatory cardiac hypertrophy to heart failure. To verify this, aortic banding (AB) was created to mimic pressure overload in wild-type (WT) mice, which were treated with sodium hydrosulfide (NaHS, H₂S donor) in drinking water and compared with untreated control mice. Mice were studied at 3 and 8 wk. In the NaHS-treated AB 8 wk group, the expression of MMP-2, CD31, and VEGF was increased while the expression of MMP-9, endostatin, angiostatin, and tissue inhibitor of matrix metalloproteinase (TIMP)-3 was decreased compared with untreated control mice. There was significant reduction in fibrosis in NaHS-treated groups. Echocardiograph and pressure-volume data revealed improvement of cardiac function in NaHS-treated groups over untreated controls. These results show that H₂S by inducing MMP-2 promotes VEGF synthesis and angiogenesis while it suppresses MMP-9 and TIMP-3 levels, inhibits antiangiogenic factors, reduces intracardiac fibrosis, and mitigates transition from compensatory hypertrophy to heart failure.

Activation of GABA-A Receptor Ameliorates Homocysteine-Induced MMP-9 Activation by ERK Pathway

Hyperhomocysteinemia (HHcy) is a risk factor for neuroinflammatory and neurodegenerative diseases. Homocysteine (Hcy) induces redox stress, in part, by activating matrix metalloproteinase‐9 (MMP‐9), which degrades the matrix and leads to blood–brain barrier dysfunction. Hcy competitively binds to γ‐aminbutyric acid (GABA) receptors, which are excitatory neurotransmitter receptors. However, the role of GABA‐A receptor in Hcy‐induced cerebrovascular remodeling is not clear. We hypothesized that Hcy causes cerebrovascular remodeling by increasing redox stress and MMP‐9 activity via the extracellular signal‐regulated kinase (ERK) signaling pathway and by inhibition of GABA‐A receptors, thus behaving as an inhibitory neurotransmitter. Hcy‐induced reactive oxygen species production was detected using the fluorescent probe, 2′–7′‐dichlorodihydrofluorescein diacetate. Hcy increased nicotinamide adenine dinucleotide phosphate‐oxidase‐4 concomitantly suppressing thioredoxin. Hcy caused activation of MMP‐9, measured by gelatin zymography. The GABA‐A receptor agonist, muscimol ameliorated the Hcy‐mediated MMP‐9 activation. In parallel, Hcy caused phosphorylation of ERK and selectively decreased levels of tissue inhibitors of metalloproteinase‐4 (TIMP‐4). Treatment of the endothelial cell with muscimol restored the levels of TIMP‐4 to the levels in control group. Hcy induced expression of iNOS and decreased eNOS expression, which lead to a decreased NO bioavailability. Furthermore muscimol attenuated Hcy‐induced MMP‐9 via ERK signaling pathway. These results suggest that Hcy competes with GABA‐A receptors, inducing the oxidative stress transduction pathway and leading to ERK activation. J. Cell. Physiol. 220: 257–266, 2009.

Cardiac specific deletion of N-methyl-D-aspartate receptor 1 ameliorates mtMMP-9 mediated autophagy/mitophagy in hyperhomocysteinemia

Autophagy is an important process in the pathogenesis of cardiovascular diseases; however, the proximal triggers for mitochondrial autophagy were unknown. The N-methyl-d-aspartate receptor 1 (NMDA-R1) is a receptor for homocysteine (Hcy) and plays a key role in cardiac dysfunction. Cardiac-specific deletion of NMDA-R1 has been shown to ameliorate Hcy-induced myocyte contractility. Hcy activates mitochondrial matrix metalloproteinase-9 (mtMMP-9) and induces translocation of connexin-43 (Cxn-43) to the mitochondria (mtCxn-43). We sought to show cardiac-specific deletion of NMDA-R1 mitigates Hcy-induced mtCxn-43 translocation, mtMMP-9-mediated mtCxn-43 degradation, leading to mitophagy, in part, by decreasing mitochondrial permeability (MPT). Cardiac-specific knockout (KO) of NAMDA-R1 was generated using the cre/lox approach. The myocyte mitochondria were isolated from wild type (WT), WT + Hcy (1.8 g of DL-Hcy/L in the drinking water for 6 weeks), NMDA-R1 KO + Hcy, and NR1fl/fl/Cre (NR1fl/fl) genetic control mice. Mitochondrial respiratory capacity and MPT were measured by fluorescence-dye methods. The mitochondrial superoxide and peroxinitrite levels were detected by confocal microscopy using Mito-SOX and dihydrorhodamine-123. The mtMMP-9 activity and expression were detected by zymography and RT-PCR analyses. The mtCxn-43 translocation was detected by confocal microscopy. The degradation of mtCxn-43 and LC3-I/II (a marker of autophagy) were detected by Western blot. These results suggested that Hcy enhanced intramitochondrial nitrosative stress in myocytes. There was a robust increase in mtMMP-9 activity. An increase in translocation and degradation of mtCxn-43 was also noted. These increases led to mitophagy. The effects were ameliorated by cardiac-specific deletion of NMDA-R1. We concluded that HHcy increased mitochondrial nitrosative stress, thereby activating mtMMP-9 and inciting the degradation of mtCxn-43. This led to mitophagy, in part, by activating NMDA-R1. The findings of this study will lead to therapeutic ramifications for mitigating cardiovascular diseases by inhibiting the mitochondrial mitophagy and NMDA-R1 receptor.

Homocysteine mediated decrease in bone blood flow and remodeling: role of folic acid.

Deficiencies in folate lead to increased serum concentrations of homocysteine (Hcy), which is known as hyperhomocysteinemia (HHcy), is associated with bone disorders. Although, Hcy accumulates collagen in bone and contribute to decrease in bone strength. The mechanism of Hcy induced bone loss and remodeling is unclear. Therefore, the present study was aimed to determine the role of folic acid (FA) in genetically HHcy‐associated decrease in bone blood flow and remodeling. Wild type (WT) and cystathionine‐β‐synthase heterozygous (CBS+/−) mice were used in this study and supplemented with or without FA (300 mg/kg, Hcy reducing agent) in drinking water for 6 weeks. The tibial bone blood flow was measured by laser Doppler and ultrasonic flow probe method. The tibial bone density (BD) was assessed by dual energy X‐ray absorptiometry. The bone homogenates were analyzed for oxidative stress, NOX‐4 as oxidative marker and thioredoxin‐1 (Trx‐1) as anti‐oxidant marker, bone remodeling (MMP‐9) and bio‐availability of nitric oxide (eNOS/iNOS/NO) by Western blot method. The results suggested that there was decrease in tibial blood flow in CBS+/− mice. The BD was also reduced in CBS+/− mice. There was an increase in NOX‐4, iNOS, MMP‐9 protein as well as MMP‐9 activity in CBS+/− mice and decrease in Trx‐1, eNOS protein levels, in part by decreasing NO bio‐availability in CBS+/− mice. Interestingly, these effects were ameliorated by FA and suggested that FA supplementation may have therapeutic potential against genetically HHcy induced bone loss.

Hydrogen sulfide mitigates matrix metalloproteinase-9 activity and neurovascular permeability in hyperhomocysteinemic mice

An elevated level of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), was associated with neurovascular diseases. At physiological levels, hydrogen sulfide (H(2)S) protected the neurovascular system. Because Hcy was also a precursor of hydrogen sulfide (H(2)S), we sought to test whether the H(2)S protected the brain during HHcy. Cystathionine-beta-synthase heterozygous (CBS+/-) and wild type (WT) mice were supplemented with or without NaHS (30 microM/L, H(2)S donor) in drinking water. Blood flow and cerebral microvascular permeability in pial vessels were measured by intravital microscopy in WT, WT+NaHS, CBS-/+ and (CBS-/+)+NaHS-treated mice. The brain tissues were analyzed for matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) by Western blot and RT-PCR. The mRNA levels of CBS and cystathionine gamma lyase (CSE, enzyme responsible for conversion of Hcy to H(2)S) genes were measured by RT-PCR. The results showed a significant increase in MMP-2, MMP-9, TIMP-3 protein and mRNA in CBS (-/+) mice, while H(2)S treatment mitigated this increase. Interstitial localization of MMPs was also apparent through immunohistochemistry. A decrease in protein and mRNA expression of TIMP-4 was observed in CBS (-/+) mice. Microscopy data revealed increase in permeability in CBS (-/+) mice. These effects were ameliorated by H(2)S and suggested that physiological levels of H(2)S supplementation may have therapeutic potential against HHcy-induced microvascular permeability, in part, by normalizing the MMP/TIMP ratio in the brain.

Mitochondrial mitophagic mechanisms of myocardial matrix metabolism and remodelling

High levels of homocysteine (Hcy), known as hyperhomocysteinmia (HHcy), are correlated with an increase in extracellular matrix remodelling (ECM) via the matrix metalloproteinases (MMPs) and plasminogen/plasmin system. This results in an increase deposition of collagen that leads to endothelial-myocyte (EM) and myocyte-myocyte (MM) uncoupling; the physiological consequences are a plethora of cardiovascular pathologies. Homocysteine-induced increase in intracellular and mitochondrial Ca2+ plays an important role in increasing reactive oxygen species (ROS) within mitochondria and instigating mitophagy within the cell. This occurs via several Hcy-mitigated processes: agonizing N-methyl-d-aspartate receptor-1 (NMDA-R1), decreasing expression of peroxisome proliferator activator receptor (PPAR) [thereby increasing oxidation], impairing Ca2+ handling via Na+/Ca2+ exchanger (NCX1) and Sarco endoplasmic reticulum Ca2+ ATPase (SERCA-2a). The end result is an increase in ROS that directly or indirectly lead to MMP activation within mitochondria or the cytoplasm. Hcy induces a mitochondrial permeability transition that allows MMPs to be released from mitochondria thereby metabolizing matrix and impairing cardiac function. Further work remains to be elucidated concerning the specific mitochondrial mitophagic mechanisms under which matrix metabolism and remodelling occurs. Moreover, the therapeutic implications of NMDA and PPAR ligands are some promise to patient.

Hyperhomocysteinemia decreases bone blood flow

Elevated plasma levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), are associated with osteoporosis. A decrease in bone blood flow is a potential cause of compromised bone mechanical properties. Therefore, we hypothesized that HHcy decreases bone blood flow and biomechanical properties. To test this hypothesis, male Sprague–Dawley rats were treated with Hcy (0.67 g/L) in drinking water for 8 weeks. Age-matched rats served as controls. At the end of the treatment period, the rats were anesthetized. Blood samples were collected from experimental or control rats. Biochemical turnover markers (body weight, Hcy, vitamin B12, and folate) were measured. Systolic blood pressure was measured from the right carotid artery. Tibia blood flow was measured by laser Doppler flow probe. The results indicated that Hcy levels were significantly higher in the Hcy-treated group than in control rats, whereas vitamin B12 levels were lower in the Hcy-treated group compared with control rats. There was no significant difference in folate concentration and blood pressure in Hcy-treated versus control rats. The tibial blood flow index of the control group was significantly higher (0.78 ± 0.09 flow unit) compared with the Hcy-treated group (0.51 ± 0.09). The tibial mass was 1.1 ± 0.1 g in the control group and 0.9 ± 0.1 in the Hcy-treated group. The tibia bone density was unchanged in Hcy-treated rats. These results suggest that Hcy causes a reduction in bone blood flow, which contributes to compromised bone biomechanical properties.

Electrical stimulation of cardiomyocytes activates mitochondrial matrix metalloproteinase causing electrical remodeling.

Cardiac arrhythmias, instigated by mechanical and electrical remodeling, are associated with activation of extracellular matrix metalloproteinases (MMPs). However, the connection between intracellular MMPs activation and arrhythmogenesis is not well established. Previously, we determined localization of MMP in the mitochondria using confocal microscopy. We tested the hypothesis that electrical pacing induces the activation of mitochondrial MMP (mtMMP) and is associated with myocyte mechanical dysfunction. Myocytes were isolated and field stimulated at 1 and 4 Hz. Myocyte mechanics and calcium transient was studied using Ion-Optix system. Mitochondrial MMP-9 activation was evaluated using zymography. There was a 25% increase in 1 Hz and 40% increase in 4 Hz stimulation. We observed an increase in mtMMP activation with increase in electrical pacing compared to 0 Hz with a significant increase (p<0.05, n=3). Field stimulation at 4 Hz decreased cell re-lengthening. The levels of calcium transient were reduced with increase in contraction frequency. We conclude that electrical stimulation activates mtMMP-9 that is associated with myocyte mechanical dysfunction.

Differential expression of Gs in a murine model of homocysteinemic heart failure.

High plasma homocysteine levels are a known risk factor in heart failure and sudden cardiac death. The G proteins, Gs (stimulatory) and Gi (inhibitory), are involved in calcium regulation; overexpression has pathological consequences. The aims of this study were to examine the differential expression of Gs G protein and Gi in the hearts of hyperhomocysteinemic (Hhcy) mice, and to determine if homocysteine (Hcy) acts as an agonist in cell culture to mediate the change in G protein isoforms. To create Hhcy, heterozygous cystathionine-β-synthase (CBS) knockout (KO) mice were used. Mice were sacrificed, hearts were excised, cardiac tissue homogenates were prepared, and Western blots were performed. The results suggested that Gs G protein was downregulated in cardiac tissue of heterozygous CBS KO mice to 46% that of control hearts. However, the intracellular Gi G protein content remained the same in heterozygous CBS KO mice. Transformed cardiomyocyte HL-1 cells were treated with varying concentrations of homocysteine. The results suggested no detectable differential Gs and Gi expression. This suggested that Hcy did not act as an agonist in vitro to alter G protein content, but that Hcy produced some other in vivo effects to incur these results.