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circular rnas ( circrnas ) are involved in the development of various diseases ; however , knowledge on circrnas in osteoarthritis ( oa ) is limited .
this study aims to identify circrna expression in different regions affected by oa and to explore the function of mechanical stress - related circrnas ( circrnas - msr ) in cartilage .
bioinformatics was employed to predict the interaction of circrnas and mrnas in the cartilage .
loss - of - function experiments for circrnas - msr were performed in vitro .
a total of 104 circrnas were differentially expressed in damaged versus intact cartilage . of these circrnas , 44 and 60 were upregulated and downregulated , respectively , in the damaged tissue .
circrna - msr expression increased under mechanical stress in chondrocytes .
circrnas - msr were silenced using small interfering rna , and knockdown of circrnas - msr could suppress tumor necrosis factor alpha ( tnf- ) expression and increase extracellular matrix ( ecm ) formation .
our results demonstrated that circrnas - msr regulated tnf- expression and participated in the chondrocyte ecm degradation process .
we propose that the inhibition of circrnas - msr could inhibit the degradation of chondrocyte ecm and knockdown of circrnas - msr could be a potential therapeutic target for oa . . osteoarthritis ( oa ) is a degenerative joint disease characterized by articular cartilage degradation , subchondral bone thickening , and osteophyte formation.1 , 2 cartilage cellularity is reduced in oa through chondrocyte death , in which chondrocytes are stimulated by cytokines and growth factors to undergo catabolic and abnormal differentiation , leading to extracellular matrix ( ecm ) degradation.3 , 4 , 5 , 6 in oa , the medial compartment of the articular cartilage is the most susceptible to degeneration , whereas the lateral compartment remains relatively unaffected.7 , 8 differences in mechanical factors underlie the disparity in disease susceptibility between the medial and lateral compartments ; the damaged cartilage regions are usually subjected to mechanical loading , whereas the intact regions are not .
the chondrocytes in the damaged cartilage are particularly susceptible to mechanical stress , and the tensile properties of the damaged cartilage are lost because of the destructed collagen network.9 , 10 circular rnas ( circrnas ) are a large class of noncoding rnas that exist ubiquitously in the cytoplasm of eukaryotic cells;11 , 12 these endogenous rnas are characterized by stable structure and high tissue - specific expression . compared with linear rnas , circrnas can remarkably undergo non - canonical splicing without a free 3 or 5 end.14 , 15 recent reports show that circrnas function as microrna ( mirna ) sponges that naturally sequester and competitively suppress mirna activity .
the involvement of circrnas is demonstrated in the development of several types of diseases , such as atherosclerosis and nervous system disorders.17 , 18 , 19 however , the role of circrnas in cartilage and their overall contribution to oa pathogenesis are still unknown .
the present study identified a small number of up- or downregulated circrnas in different cartilage regions .
we specifically identified a new circrna , called mechanical stress - related circrna ( circrna - msr ) , involved in mechanical stress .
we demonstrate that circrna - msr controls tumor necrosis factor alpha ( tnf- ) expression and promotes the degradation of ecm .
the cartilage was assessed by histologic examination , and the histologic scores were graded according to a modified mankin scale .
the score of < 4 points was considered intact cartilage and a score of > 6 represented damaged cartilage .
hierarchical clustering revealed the circrna expression in the cartilage samples ( figure 1a ) .
the scatter and volcano plots showed varied circrna expressions between the damaged and intact cartilage samples ( figures 1b and 1c ) .
we identified 104 differentially expressed circrnas in the damaged cartilage compared to the intact cartilage .
of these circrnas , 44 were upregulated and 60 were downregulated in the damaged tissue samples ( table s1 ) . to validate the circrna microarray results , we performed qpcr to analyze the changes in expression among the differentially expressed circrnas .
the data confirmed that circrna_000598 , circrna_103387 , circrna_101975 , and circrna_100226 ( circrnas - msr ) were overexpressed in the damaged region compared with the intact region of the cartilage in oa ( figure 2a ) .
a total of 89 circrnas and 61 mrnas were noted in the damaged cartilage network , whereas 94 circrnas and 36 mrnas were in the intact cartilage network . in these co - expression networks ,
circrnas and mrnas were connected by a string , indicating a tight correlation between these genes and a potential regulatory relationship ( figures s1 and s2 ) .
furthermore , we constructed another network with circrnas , mrnas , and the common binding mirnas .
the differentially expressed circrnas were annotated in detail with the mirna interaction information ( table s1 ) .
we selected cartilage - specific mrnas according to gene ontology ( go ) and pathway analyses ( figures 2b and 2c ) .
we constructed a network of circrnas - mirnas - mrnas , with a total of 45 circrnas , 42 mrnas , and 42 mirnas , by merging the commonly targeted mirnas of circrnas and mrnas ( figure 3 ) .
this indicated the tight correlation and regulation relationship of these genes in the network .
we assumed that circrna - msr was a special cartilage circrna ( hsa_circ_100226 , i d circ_0005567 in circbase ; http://circbase.org ) , with its gene located at chr1:51868106 - 51874004 and the symbol of the associated gene being eps15 .
the circrna - msr was chosen because it is one of the circrnas indicated to be associated with mechanical stress and inflammatory response according to the bioinformatics analysis mentioned above . to investigate the effect of mechanical stress on chondrocytes , the cells were exposed to pre - optimized magnitudes of cyclic tensile strain ( cts ) using flexcell-5000 ( figure 4a ) .
the expression levels of col2a1 and aggrecan were significantly downregulated , whereas the expression levels of tnf- and il-1 were significantly upregulated by cts for 24 hr ( figure 4b ) .
moreover , we found that circrna - msr expression paralleled tnf- expression under mechanical stress ( figure 4c ) .
therefore , we speculated the co - regulated expression of circrna - msr and tnf- under mechanical stress in vitro conditions . to analyze the effects of circrna - msr on tnf- expression in chondrocyte ecm degradation , we examined the effect of circrna - msr knockdown on damaged chondrocytes .
the small interfering rna ( sirna ) used in this experiment was specific for circrna - msr .
as a consequence of this inhibition , the mrna expression for tnf- decreased , whereas the ecm expression significantly rose by si - msr treatment ( figure 5a ) .
the immunofluorescence results also showed that ecm proteins were significantly elevated by the si - msr treatment ( figures 5b and 5c ) .
circrnas have been reported to function as mirna sponges that naturally sequester and competitively suppress mirna activity .
we assumed that circrna - msr functions as a decoy to regulate tnf- expression through the same mechanism . according to the informatics analysis
, there were five mirna - binding sites for circrna - msr , and they were mir-138 , mir-145 , mir-24 , mir-620 , and mir-875 ( figure 6a ) .
the circrna - msr 3 utr sequence matched these mirnas , and the tnf- 3 utr matched mir-875 ( figure 6b ) .
thus , we identified a common mirna ( mir-875 ) for circrna - msr and tnf- targets .
oa is a multifactorial disease characterized by progressive inflammation , pain , and cartilage destruction in load - bearing surfaces of the knee joints . among various pathogenic factors ,
aberrant mechanical stimulation results in a physiological imbalance between mechanical stress on the joint and the joint s ability to withstand such stress .
chondrocytes are directly exposed to compression force during cartilage loading ; thus , the ecm of chondrocytes tends to stretch the cells during cartilage compression .
many studies on oa focused on the epigenetic regulation of its pathogenesis and potential targets for therapy , including mirnas and long noncoding rnas ( lncrnas ) . however , the occurrence of circrnas in cartilage remains largely unknown .
this study identified a number of aberrantly expressed circrnas in damaged regions compared with the intact regions of oa .
we found that circrna - msr is vital under mechanical stress in loss - of - function experiments and that it regulates the tnf- in the process of ecm degradation in chondrocytes .
recent studies showed that numerous exonic transcripts can form circrnas through non - linear reverse splicing or gene rearrangement .
the two properties of circrnas are the most important : first , they are highly conserved sequences ; second , they show a high degree of stability in mammalian cells . compared with other noncoding rnas , such as mirnas and lncrnas ,
these properties provide circrnas with the potential to be used as ideal biomarkers and potential therapy targets .
we assumed that circrna - msr functioned as a decoy to regulate tnf- expression through the same mechanism .
we found that circrna - msr harbors mirna - binding sites , including mir-138 , mir-145 , mir-24 , mir-620 , and mir-875 .
in addition , mir-875 can bind to the 3 utr of tnf-. however , this study focused on mir-875 as the only mirna that can target both circrna - msr and tnf-. this study demonstrated that circrna - msr regulated tnf- expression and participated in the chondrocyte ecm degradation process .
we confirmed that the silencing of circrna - msr by sirna can suppress tnf- expression and increase ecm formation .
thus , circrna - msr can be used as a potential target and specific sirnas can be used as therapeutic agents in oa therapy .
the most attractive aspect of this therapeutic is the ability to target the gene(s ) , which may not be possible with small molecules or protein - based drugs .
this opens up a whole new therapeutic approach for the treatment of oa by targeting genes that are causally involved in the pathological process .
although this approach is promising , several challenges have been identified , including the lack of stability against extracellular and intracellular degradation by nucleases , poor uptake and low potency at target sites of sirnas , and off - target effects .
collectively , our data indicate that 104 circrnas were either over- or underexpressed in oa .
the observed changes were suggested to have biologic effects and that circrnas are key regulators of gene expression .
we confirmed that circrna - msr is the decoy for tnf-. the mechanism needs to be confirmed with further specific studies .
deciphering the precise molecular mechanisms of circrna function in oa is critical to understanding oa pathogenesis and exploring new potential therapeutic targets .
oa cartilage was isolated from the knee joints of 30 patients undergoing total knee arthroplasty .
joint tissue was immediately shock - frozen in liquid nitrogen , and the articular cartilage was isolated within 24 hr from the condyles and tibia plateaus with the use of a surgical blade .
the study was approved by the human ethics committee of the peking university third hospital ( china ) .
the cartilage was removed and fixed in 4% paraformaldehyde in pbs solution ( ph 7.4 ) for 48 hr at 4c , and then it was demineralized in 15% edta ( ph 7.2 ) in pbs for 2 weeks .
the cartilage specimens were dehydrated in a graded series of alcohol and xylene , and then they were embedded in paraffin and cut serially into 5 m sagittal sections . the sections were stained with toluidine blue , safranin - o , and h&e as per routine protocol .
scores of < 4 and > 6 points indicated intact and damaged cartilage , respectively .
joint tissue was immediately shock - frozen in liquid nitrogen , and then the articular cartilage was isolated from the condyles and tibia plateaus using a surgical blade within 24 hr .
afterward , the samples were homogenized in trizol reagent ( invitrogen ) , and the total rna in each sample was quantified using a nanodrop nd-1000 . sample preparation and microarray hybridization
the total rna from each sample was amplified and transcribed into fluorescent crna utilizing random primers according to the arraystar super rna labeling protocol .
the labeled crnas were hybridized onto the arraystar human circrna array ( 8 15 k ) .
we selected specific mrnas to construct the network according to our previous microarray data and the enrichment analyses of go .
co - expression networks were constructed according to the normalized signal intensities of circrnas and mrnas in the original microarray data .
pearson s correlation analysis was applied to measure the significance of the correlation of the expressions between each gene pair .
when the expression levels of two genes were similar above a preselected threshold in the pearson analysis , they were considered to exhibit a co - expression relationship and would be connected .
each gene corresponded to a node , and two genes were connected by a string , indicating a tight correlation .
the degree of correlation determined gene importance in the network.30 , 31 an mrna - mirna - circrna network was constructed according to the common target mirnas of the circrnas and mrnas .
the interactions of circrnas and mrnas with mirnas were predicted with the arraystar mirna target prediction software based on targetscan and miranda.32 , 33 donor chondrocytes were isolated as previously described .
chondrocytes ( 5 105/well ) were at passage 2 and grown on pronectin f - coated bioflex six - well culture plates ( flexcell international ) to 80% confluence .
our previous study selected the pre - optimized magnitudes of cts , so the chondrocytes were enforced at 10% elongation ( 0.5 hz ) for the indicated times.34 , 35 the sirnas targeting circrna - msr ( referred to as si - circmsr ) were designed and synthesized by ribobio .
chondrocytes were transfected with si - circmsr using lipofectamine 3000 ( invitrogen ) according to the manufacturer s protocol .
prior to transfection , the culture medium was replaced with a medium without antibiotics , and the cells were cultured for 24 hr ( cyagen biosciences ) .
the rnai lipofectamine 3000 complex was prepared by mixing for 20 min , and the complex was then added to each cell .
total rna was isolated from cartilage tissues or monolayer - cultured primary chondrocytes using trizol reagent .
homogenized tissue samples were in 1 ml trizol reagent per 50100 mg , and the lysed cells were directly added to 1 ml trizol reagent in a 3.5-cm diameter dish . for mirna qpcr analysis , reverse transcription of specific mirnas was performed with the bulge - loop mirna primer set ( ribobio ) according to the manufacturer s instructions . for mrna analysis ,
the mrna expression levels were reported relative to glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) , whereas mirna expression levels were reported relative to u6 .
the primers used in the present study are as follows : tnf- forward : 5-cctctctctaatcagccctctg-3 , reverse : 5-gaggacctgggagtagatgag-3;circrna - msr forward : 5-tccagtctgatccttttgttgg-3 , reverse : 5-ctgtttcttgctgtagacggct-3;hsa_circrna_103387 forward : 5-agtctttccaccttggctct-3 , reverse : 5-tggacagggtacttctcgttt-3;hsa_circrna_000598 forward : 5-gtcccttccctgtcactacct-3 , reverse : 5-tctgttgatgccgccttgg-3;hsa_circrna_101975 forward : 5-gcccaaaccagacctcactt-3 , reverse : 5-tccttctcgggctcctga-3 ; andgapdh forward : 5-gggaaactgtggcgtgat-3 , reverse : 5-gagtgggtgtcgctgttga-3. tnf- forward : 5-cctctctctaatcagccctctg-3 , reverse : 5-gaggacctgggagtagatgag-3 ; circrna - msr forward : 5-tccagtctgatccttttgttgg-3 , reverse : 5-ctgtttcttgctgtagacggct-3 ; hsa_circrna_103387 forward : 5-agtctttccaccttggctct-3 , reverse : 5-tggacagggtacttctcgttt-3 ; hsa_circrna_000598 forward : 5-gtcccttccctgtcactacct-3 , reverse : 5-tctgttgatgccgccttgg-3 ; hsa_circrna_101975 forward : 5-gcccaaaccagacctcactt-3 , reverse : 5-tccttctcgggctcctga-3 ; and gapdh forward : 5-gggaaactgtggcgtgat-3 , reverse : 5-gagtgggtgtcgctgttga-3. the cultured cells were rinsed in pbs and fixed with 4% paraformaldehyde for 15 min at room temperature .
the cultured cells were incubated with anti - col2 ( 1:200 dilution ) and aggrecan ( 1:200 dilution ) at 4c overnight .
the cells were subsequently incubated for 1 hr with fluorescein isothiocyanate - conjugated affinipure goat anti - rabbit igg ( 1:100 dilution ) .
finally , the samples were incubated for 5 min with hoechst 33342 and observed with a confocal microscope ( fv 1000 olympus ix-81 ) .
the results are reported as the mean sem ; p < 0.05 was considered statistically significant .
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abstractaims / introduction : angiotensin ii type 1 receptor blockers ( arb ) are regarded as firstline treatment for type 2 diabetes with hypertension . however
, lowering blood pressure to the target level often requires more than one antihypertensive agent as recommended by the guideline . in this openlabel , prospective , crossover clinical trial , we compared the effects of combination treatment of arb with a calcium channel blocker ( ccb ) or with a lowdose thiazide diuretic on blood pressure ( bp ) and various metabolic parameters in hypertensive patients with type 2 diabetes.materials and methods : a total of 39 japanese type 2 diabetics with hypertension treated with olmesartan ( 20 mg / day ) for at least 8 weeks were recruited to this study . at study entry ,
treatment was switched to either olmesartan ( 20 mg / day)/azelnidipine ( 16 mg / day ) or olmesartan ( 20 mg / day)/trichlormethiazide ( 1 mg / day ) and continued for 12 weeks .
then , the drugs were switched and treatment was continued for another 12 weeks .
we measured clinical blood pressure and various metabolic parameters before and at the end of each study arm.results : compared with the olmesartan / trichlormethiazide treatment , treatment with olmesartan / azelnidipine achieved superior clinical blood pressure and pulse rate control .
in contrast , the treatment with olmesartan / trichlormethiazide resulted in increased hba1c , serum uric acid and worsening of estimated glomerular filtration rate , though there were no differences in other metabolic parameters including urine 8hydroxy2deoxyguanosine , creactive protein and adiponectin between the two treatments.conclusions : our results show that the combination of arb with azelnidipine is more beneficial with regard to blood pressure control and metabolic outcome than the combination of olmesartan with low dose trichlormethiazide .
this trial was registered with umin clinical trial registry ( no .
umin000005064 ) .
( j diabetes invest , doi : 10.1111/j.20401124.2011.00135.x , 2011 ) . patients with type 2 diabetes are at high risk of developing cardiovascular diseases , which are also the most common causes of death in these patients . in type 2
diabetes , the prevalence of hypertension is higher than the general population , and hypertension associated with diabetes increases the incidence of cardiovascular disease .
thus , treatment of hypertension in addition to glycemic control is important in order to reduce cardiovascular events in patients with type 2 diabetes , as it was emphasized in the seventh report of the joint national committee on prevention , detection , evaluation , and treatment of high blood pressure ( jnc7 ) . according to that report
, the target clinical arterial blood pressure for hypertensive patients with diabetes mellitus is < 130/80 mmhg and the use of blockers of the renin
angiotensin system ( ras ) , either angiotensin ii type i receptor blockers ( arb ) or angiotensinconverting enzyme ( ace ) inhibitors , are recommended as the primary antihypertensive drugs .
the wide use of arb in the treatment of hypertension is based on their beneficial effects on hypertensionrelated cardiovascular endorgan damage , at least in part , through reduction of oxidative stress and inflammation in addition to their blood pressure lowering effects . in particular , among the clinically available arb , olmesartan has potent blood pressure lowering effects at a regular dose , compared with other agents . however , in the majority of patients , the use of one kind of firstline antihypertensive agent is insufficient to achieve a strict target blood pressure level , and a combination of multiple antihypertensive drugs is required to induce an adequate fall in blood pressure .
generally , arb or ace inhibitors are often used in combination with a small dose of thiazide diuretics or calcium channel blockers ( ccb ) .
thiazide diuretics are beneficial for patients with hypertension , as they reduce cardiovascular morbidity and mortality .
in addition , they are commonly used in combination with arb or ace inhibitors , as they promote na excretion and hence enhance the blood lowering effects of arb or ace inhibitors .
however , thiazide diuretics are known to cause various metabolic abnormalities , such as insulin resistance , newonset diabetes mellitus , hypokalemia and hyperuricemia . in contrast , ccb can also reduce cardiovascular events in patients with coronary disease or highrisk hypertensives .
furthermore , the combination of ccb with arb or ace inhibitors effectively lowers blood pressure without any metabolic adverse effects . the recent accomplish ( avoiding cardiovascular events through combination therapy in patients living with systolic hypertension ) trial compared the relative merits of the combination of ace inhibitors with those two types of antihypertensive agents .
the results showed that the combination of ccb with ace inhibitors is more effective in reducing cardiovascular events compared to the combination of thiazide diuretics with ace inhibitors in highrisk hypertensive patients .
however , the effects of arb combined with ccb or thiazide diuretics have not been fully evaluated in type 2 diabetic patients with hypertension . the present study is an open label crossover trial in hypertensive type 2 diabetic patients treated with a combination of arb ( olmesartan 20 mg / day ) and azelnidipine or lowdose thiazide diuretic ( trichlormethiazide 1 mg / day ) .
blood pressure , pulse rate , various metabolic parameters and renal function were assessed throughout the study .
all patients with type 2 diabetes mellitus who visited juntendo university hospital ( tokyo , japan ) , juntendo tokyo koto geriatric medical center ( tokyo , japan ) , juntendo university urayasu hospital ( urayasu , japan ) , juntendo university sizuoka hospital ( shizuoka , japan ) , and juntendo university nerima hospital ( tokyo , japan ) between january 2008 and march 2010 were invited to participate in the study .
the inclusion criteria were patients with type 2 diabetes mellitus and hypertension in whom the target clinic blood pressure ( < 130/80 mmhg ) could not be achieved despite treatment with olmesartan 20 mg once daily for at least 8 weeks .
in addition , patients with severe renal ( estimated glomerular filtration rate [ egfr ] < 30 ) or hepatic disease , overt cardiovascular disease and malignancy were excluded .
diabetic nephropathy was defined as an albumin to creatinine ratio 30 mg / g creatinine by examination of a spot urine sample .
diabetic neuropathy was defined by the recommendation of the japan diabetes society ( jds ) using sensory symptoms in the bilateral lower limbs including tingling , pain , allodynia or unusual sensations , or bilateral absence of the achilles tendon reflex , or in case of diminished sensitivity .
the ethics committees of the participating hospitals approved the study protocol and informed consent was obtained from each subject .
an open label crossover design was applied to the present study . at the end of the monotherapy of olmesartan 20 mg oncedaily as the antihypertensive drug , blood pressure
was measured and fasting blood samples were collected as baseline data . then , the participants were randomized into one of two treatment groups who received either 16 mg azelnidipine or 1 mg trichlormethiazide once daily in the morning in addition to olmesartan 20 mg / day .
after 12 weeks of azelnidipine / olmesartan treatment , blood pressure was again measured and fasting blood samples were collected . then , the subjects on azelnidipine were switched to trichlormethiazide , whereas the subjects on trichlormethiazide were switched to azelnidipine , and each continued the treatment for another 12 weeks , after which blood pressure was measured with fasting blood sampling ( figure 1 ) .
blood pressure and pulse rate were recorded at the outpatient clinic , and the reported values represented the average of triplicate measurements taken at intervals of 1 min with the cuff on the left arm in a sitting position after a 5min rest .
all subjects were advised to consume their usual diet and exercise during the study period .
each patient was reviewed as to their general health and compliance with the medication , which was assessed by tablet counts and checking of blood pressure , bodyweight , and diet and exercise status at each visit . during the study period ,
schematic diagram of the study protocol . blood sampling and blood pressure ( bp ) measurement
blood samples obtained at week 12 and 24 were used for evaluation of the effects of each drug .
serum lipids ( highdensity lipoprotein cholesterol , lowdensity lipoprotein cholesterol and triglycerides ) , glucose , hba1c , creatinine , uric acid , sodium and potassium were measured with standard techniques .
the value for hba1c ( % ) is estimated as a national glycohemoglobin standardization program ( ngsp ) equivalent value ( % ) calculated by the formula hba1c ( % ) = hba1c ( jds ) ( % ) + 0.4% , considering the relational expression of hba1c ( jds ) ( % ) measured by the previous japanese standard substance and measurement methods and hba1c ( ngsp ) .
measurement of highly sensitive creactive protein ( hscrp ) by latex nephelometry was outsourced to a private laboratory ( srl laboratory , tokyo , japan ) . homeostasis model assessment of insulin resistance ( homair ) represented the product of fasting plasma insulin ( u / ml ) and fasting plasma glucose levels ( mmol / l ) divided by 22.5 .
the egfr was calculated by the formula for japanese patients : egfr ( ml / min per 1.73 m ) = 194 age serum creatinine ( 0.739 for females ) .
urinary 8hydroxy2deoxyguanosine ( 8ohdg ) was measured by enzymelinked immunosorbent assay ( elisa ) using a spot urine sample ( srl laboratory ) , and the results are expressed relative to creatinine ( per mg cr ) .
results are presented as mean sd or median ( range 2575% ) .
differences between groups were examined for statistical significance using the twotailed paired student s ttest or wilcoxon signedrank test when data did not show normal distribution .
all patients with type 2 diabetes mellitus who visited juntendo university hospital ( tokyo , japan ) , juntendo tokyo koto geriatric medical center ( tokyo , japan ) , juntendo university urayasu hospital ( urayasu , japan ) , juntendo university sizuoka hospital ( shizuoka , japan ) , and juntendo university nerima hospital ( tokyo , japan ) between january 2008 and march 2010 were invited to participate in the study .
the inclusion criteria were patients with type 2 diabetes mellitus and hypertension in whom the target clinic blood pressure ( < 130/80 mmhg ) could not be achieved despite treatment with olmesartan 20 mg once daily for at least 8 weeks .
in addition , patients with severe renal ( estimated glomerular filtration rate [ egfr ] < 30 ) or hepatic disease , overt cardiovascular disease and malignancy were excluded .
diabetic nephropathy was defined as an albumin to creatinine ratio 30 mg / g creatinine by examination of a spot urine sample .
diabetic neuropathy was defined by the recommendation of the japan diabetes society ( jds ) using sensory symptoms in the bilateral lower limbs including tingling , pain , allodynia or unusual sensations , or bilateral absence of the achilles tendon reflex , or in case of diminished sensitivity .
the ethics committees of the participating hospitals approved the study protocol and informed consent was obtained from each subject .
an open label crossover design was applied to the present study . at the end of the monotherapy of olmesartan 20 mg oncedaily as the antihypertensive drug , blood pressure
was measured and fasting blood samples were collected as baseline data . then , the participants were randomized into one of two treatment groups who received either 16 mg azelnidipine or 1 mg trichlormethiazide once daily in the morning in addition to olmesartan 20 mg / day .
after 12 weeks of azelnidipine / olmesartan treatment , blood pressure was again measured and fasting blood samples were collected . then , the subjects on azelnidipine were switched to trichlormethiazide , whereas the subjects on trichlormethiazide were switched to azelnidipine , and each continued the treatment for another 12 weeks , after which blood pressure was measured with fasting blood sampling ( figure 1 ) .
blood pressure and pulse rate were recorded at the outpatient clinic , and the reported values represented the average of triplicate measurements taken at intervals of 1 min with the cuff on the left arm in a sitting position after a 5min rest .
all subjects were advised to consume their usual diet and exercise during the study period .
each patient was reviewed as to their general health and compliance with the medication , which was assessed by tablet counts and checking of blood pressure , bodyweight , and diet and exercise status at each visit . during the study period ,
schematic diagram of the study protocol . blood sampling and blood pressure ( bp ) measurement were carried out at week 0 for basal data .
blood samples obtained at week 12 and 24 were used for evaluation of the effects of each drug .
serum lipids ( highdensity lipoprotein cholesterol , lowdensity lipoprotein cholesterol and triglycerides ) , glucose , hba1c , creatinine , uric acid , sodium and potassium were measured with standard techniques . the value for hba1c ( % ) is estimated as a national glycohemoglobin standardization program ( ngsp ) equivalent value ( % ) calculated by the formula hba1c ( % ) = hba1c ( jds ) ( % ) + 0.4% , considering the relational expression of hba1c ( jds ) ( % ) measured by the previous japanese standard substance and measurement methods and hba1c ( ngsp ) .
measurement of highly sensitive creactive protein ( hscrp ) by latex nephelometry was outsourced to a private laboratory ( srl laboratory , tokyo , japan ) .
homeostasis model assessment of insulin resistance ( homair ) represented the product of fasting plasma insulin ( u / ml ) and fasting plasma glucose levels ( mmol / l ) divided by 22.5 .
the egfr was calculated by the formula for japanese patients : egfr ( ml / min per 1.73 m ) = 194 age serum creatinine ( 0.739 for females ) .
urinary 8hydroxy2deoxyguanosine ( 8ohdg ) was measured by enzymelinked immunosorbent assay ( elisa ) using a spot urine sample ( srl laboratory ) , and the results are expressed relative to creatinine ( per mg cr ) .
results are presented as mean sd or median ( range 2575% ) .
differences between groups were examined for statistical significance using the twotailed paired student s ttest or wilcoxon signedrank test when data did not show normal distribution .
a total of 45 diabetic patients with hypertension were randomly assigned to the azelnidipine administered first group ( n = 23 ) and trichlormethiazide administered first group ( n = 22 ) .
six patients dropped out because of loss to follow up ( n = 3 of the olmesartan / azelnidipine group and n = 2 of the olmesartan / trichlormethiazide group ) and poor compliance ( n = 1 of the olmesartan / trichlormethiazide group ) .
no serious adverse effects were observed in all study patients including the six dropout cases .
the subjects were relatively obese ( body mass index 26.0 3.9 kg / m ) and their blood glucose , blood pressure and serum lipids were well controlled . the anthropometric data under the administration of olmesartan only were compared with those under olmesartan / azelnizipine and olmesartan / trichlormethiazide . *
comparison between olmesartan / azelnidipine and olmesartan / trichlormethiazide groups by twotailed paired student s ttest .
data are mean sd or median ( range 2575% ) . plasma insulin level and homeostasis model assessment of insulin resistance ( homair ) were not measured in patients on insulin therapy ( n = 5 ) .
8ohdg , 8hydroxy2deoxyguanosine ; egfr , estimated glomerular filtration rate ; hdlc , highdensity lipoprotein cholesterol ; hscrp , highlysensitive creactive protein ; ldlc , lowdensity lipoprotein cholesterol ; ns , not significant .
* comparison between olmesartan / azelnidipine and olmesartan / trichlormethiazide groups by twotailed student s ttest or wilcoxon signedrank test . table 2 shows clinical blood pressure under treatment with each drug . blood pressure decreased after treatment with olmesartan / azelnidipine and olmesartan / trichlormethiazide . compared with olmesartan / trichlormethiazide treatment , the combination of olmesartan / azelnidipine significantly reduced systolic and diastolic blood pressures by 5.5 and 3.0 mmhg , respectively .
the pulse rate tended to be lower during the combination therapy period with olmesartan / azelnidipine than in the monotherapy period .
in addition , the pulse rate in the olmesartan / azelnidipine group was significantly lower than that in the olmesartan / trichlormethiazide group .
table 3 lists the metabolic markers at baseline and the end of each combination therapy .
lipids and serum k did not change significantly with either of the combination therapies . with regard to glucose metabolism , hba1c was significantly lower in the olmesartan / azelnidipine group than the olmesartan / trichlormethiazide group .
however , fasting blood glucose , serum insulin and homair were comparable between the two groups .
serum uric acid and serum creatinine significantly increased in the olmesartan / trichlormethiazide group , but not in the olmesartan / azelnidipine group . reflecting these changes ,
egfr was significantly increased after olmesartan / azelnidipine treatment compared with olmesartan / trichlormethiazide treatment .
in contrast , urinary 8ohdg , hscrp and adiponectin were comparable between the two treatments .
our data showed that the combination therapy of olmesartan with azelnidipine had a more potent blood pressure lowering effect without affecting the metabolic parameters compared to that with trichlormethiazide .
the present study showed that clinical systolic and diastolic blood pressures were significantly lower in the olmesartan / azelnidipine group than in the olmesartan / trichlormethiazide group by 5.5 and 3.0 mmhg , respectively .
recently , several large trials showed that strict blood pressure control could reduce the onset of cardiovascular diseases in patients with type 2 diabetes .
for example , a 4mmhg fall in diastolic blood pressure in the hot study was found to equate with a 51% decrease in the onset of cardiovascular diseases .
similarly , a 1.92mmhg decrease in systolic blood pressure in the hope study was found to equate to a 25% reduction in cardiovascular mortality in type 2 diabetic patients . considered together with the aforementioned studies , the fall in arterial pressure recorded in the present study was clinically significant and more than a subtle change .
although increasing the dose of trichlormethiazide might achieve further reduction in blood pressure , we only used a low dose of trichlormethiazide , because the adverse effects on metabolism are more common with higher doses of thiazide diuretics . in the present study ,
the pulse rate in the olmesartan / azelnidipine group was significantly lower than in the olmesartan / trichlormethiazide group , consistent with previous reports .
azelnidipine reduces the pulse rate in essential hypertension , unlike other dihydropyridine ccb such as amlodipine , because it inhibits sympathetic nerve activity .
in contrast , diuretics are likely to increase pulse rate as a result of their action of reducing plasma volume .
epidemiological studies suggest that increased pulse rate is a predictor for cardiovascular disease and a poor prognosis , thus our data point to better beneficial effects for azelnidipine compared with thiazide diuretics .
the present study showed that serum uric acid was also significantly higher in the olmesartan / trichlormethiazide group than in the olmesartan / azelnidipine group .
recent studies have shown that thiazideinduced hyperuricemia correlates with increased cardiovascular events in hypertensive patients .
thus , care should be taken regarding uric acid monitoring during thiazide treatment , even when combined with arb .
indeed , we found that hba1c levels were significantly higher in the olmesartan / trichlormethiazide group than in the olmesartan / azelnidipine group .
however , we found that the change in hba1c level correlated neither with changes in serum uric acid level nor with serum potassium level ( data not shown ) .
further studies are needed to clarify the mechanism of the adverse effects of thiazide diuretic on glucose metabolism .
we also found that renal function assessed by serum creatinine and egfr deteriorated in the olmesartan / trichlormethiazide group .
although the mechanisms responsible for thiazideassociated renal dysfunction remain unclear , certain mechanisms , such as the chronic effects of thiazides on metabolic abnormalities like hyperuricemia , hyperglycemia or volume depletion , could be considered to impair renal function .
in contrast to our findings , the guard ( the gauging albuminuria reduction with lotrel in diabetic patients with hypertension ) study showed that the treatment using ace with thiazide diuretic resulted in a greater reduction in albuminuria without thiazideassociated renal dysfunction compared with the group using ace and ccb , although the blood pressure reduction was better in the ace and ccb group .
we could not show exact reasons for these differences , because we did not evaluate the effect on albuminuria .
recent studies have shown that azelnidipine have antiinflammatory and antioxidative stress effects and can increase serum adiponectin levels .
we evaluated the effects of azelnidipine and trichlormethiazide on inflammation , oxidative stress markers and serum adiponectin .
however , our results showed no differences in these parameters between the two treatment groups .
the reason for the lack of effect of azelnidipine on metabolic parameters in the present study is not clear at present .
it is possible that a longer duration of drug treatment is required before the appearance of these unique effects of azelnidipine . in the present study ,
just nine of the olmesartan / azelnidipine group and seven of the olmesartan / trichlormethiazide group achieved the target blood pressure of < 130/80 mmhg .
in general , a combination of more than three antihypertensive drugs is needed to achieve this goal .
thus , it is important to treat hypertension more intensively with a combination of more than three antihypertensive drugs , including thiazide diuretics , to reduce both diabetic microvascular and macrovascular complications in case of poor control with a combination of arb and ccb .
the limitation of the present study is the relatively small number of patients and the crossover design . considering the practical performance with clinical patients
in addition , we compared the effect of drugs at only one point , thus timecourse changes in blood pressure and pulse rate during the combination study could not be evaluated . regarding order effects
, there were no differences between the azelnidipinetrichlormethiazide group and the trichlormethiazideazelnidipine group by twoway analysis of variance for repeated measurements . in conclusion
, our data suggested that compared with the combination of olmesartan and trichlormethiazide , that of olmesartan and azelnidipine had superior blood pressure lowering effects , as well as superior effects on glucose and uric metabolism , and renal function in patients with type 2 diabetes .
our results suggest that the combination of olmesartan / azelnidipine can be considered ideal agents to provide better cardiovascular protection than the combination of olmesartan / trichlormethiazide in type 2 diabetic patients with hypertension .
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abstracta 41-year - old woman presented with disturbance of consciousness , right hemiparesis , and symptoms of gerstmann syndrome .
she had a history of malignant melanoma resections of an ear mole and her right neck lymph nodes and parotid gland , with subsequent chemotherapy and radiotherapy .
computed tomography showed two large lesions in the right frontal and left parietal lobes surrounded by severe brain edema .
magnetic resonance images revealed that the two lesions were strongly enhanced with cystic change , and a small round lesion was located in the left head of the caudate nucleus .
( 18f ) fluoro-2-deoxyglucose positron emission tomography showed high accumulation in both lesions , and no sign of metastatic lesions except within the brain .
the two lesions were large , causing increased intracranial pressure .
simultaneous surgical resections were performed using two approaches .
the patient s neurological symptoms were greatly improved after surgery , and her karnofsky performance status improved from 20% to 90% .
she was discharged to her home almost completely free of neurological deficits .
although , simultaneous one - stage tumor resections for multiple metastatic brain tumors do not extend the survival period , they improve the quality of the patient s limited remaining life , and may be a treatment choice for young patients with well - controlled systemic disease . . the median survival rate reported for surgically treated patients with brain metastases from melanoma ranges from 5 to 22 months .
the most significant factor influencing the survival of patients with brain metastases is the number of their cerebral metastases .
surgical treatment of multiple brain metastases is only considered a palliative solution for improving the quality of life .
however , recently the performance of multiple resections of brain metastases from melanoma has led to a reconsideration of the surgery s benefits .
we report one case of multiple brain metastases from malignant melanoma with two large lesions located in the right frontal and left parietal lobes .
this is a case report of simultaneous one - stage resections of distant lesions with two approaches for multiple metastatic malignant melanoma lesions .
a 41-year - old woman had a mole in her right ear that grew noticeably , and swelling was detected in her right neck lymph nodes .
aspiration biopsy cytology of the neck lymph node was performed , and malignant melanoma was diagnosed .
she underwent an operative resection of the ear lesion , right neck lymph nodes and parotid gland with subsequent chemotherapy and radiotherapy .
her level of consciousness had deteriorated , and she was transferred to our hospital 6 months after being diagnosed with malignant melanoma . on admission ,
her glasgow coma scale score was 11 ( e2v3m6 ) , and she had a right hemiparesis .
additionally , symptoms of gerstmann syndrome such as finger agnosias , right - left disorientation , acalculia and agraphia were noted .
computed tomography showed two large lesions in the right frontal lobe and left parietal lobe surrounded with severe brain edema ( fig .
magnetic resonance ( mr ) images revealed that the two lesions were strongly enhanced with cystic change ( fig .
1b , c , f , g ) , and a small round lesion was present in the left head of the caudate nucleus ( fig .
1i ) . the subarachnoid space surrounding the left parietal lesion was also enhanced ( fig .
( 18f ) fluoro-2-deoxyglucose positron emission tomography ( ( 18f ) fdg pet ) exhibited high levels of accumulation in the two large lesions ( fig .
1d , h ) , but revealed no signs of metastatic lesions except in the brain .
a , e : computed tomography images reveal right frontal and left parietal mass lesions .
b , c , f , g : magnetic resonance ( mr ) images show right frontal and left parietal mass lesions as strongly enhanced on t1-weighted image with gadolinium ( b , f ) , and peripheral edema on the t2-weighted image ( c , g ) .
d , h : ( 18f ) fluoro-2-deoxyglucose positron emission tomography images show high accumulations in right frontal and left parietal lesions .
i , j : axial ( i ) and sagittal ( j ) of t1-weighted images with gadolinium revealed an enhanced , small mass lesion in the left head of the caudate nucleus ( i ) , and enhanced subarachnoid space surrounding the left parietal lesion ( j ) .
first , the patient was placed in a prone position . a left parietal craniotomy and a parietal lesion resection were performed .
the patient was then turned to a supine position under general anesthesia , after which a right frontal craniotomy and a frontal lesion resection were performed . both frontal and parietal lesions consisted of soft and fragile black tissues .
both lesions were resected , but the malignant melanoma tissues in the subarachnoid space were residual ( fig .
histological examinations showed typical numerous pleomorphic and hyperchromatic cells , which confirmed the diagnosis of metastatic malignant melanoma .
a , b , c : intraoperative microscopic views show the black malignant melanoma tissues spread in subarachnoid space ( a ) , the border with normal brain was not clear ( b ) , and tumor tissues of subarachnoid space were residual ( c ) .
her consciousness disturbance , right hemiparesis and symptoms of gerstmann syndrome could no longer be seen .
she was discharged to her home 10 days after the operation , almost completely free of neurological deficits .
following surgery , the patient received a course of whole brain radiation therapy consisting of 30 gy in 10 fractions , and chemotherapy at another hospital .
then she presented with pain in her extremities , but there were no signs of bone metastases detected by fdg - pet and bone scintigraphy .
mr images showed a mild enlargement of the left head of the caudate nucleus lesion , but no signs of problems around previous surgical resections ( fig .
spinal mr images revealed a small mass lesion attached to the spinal dura mater at the atlas level ( fig .
the patient presented with a generalized seizure and was again admitted to a nearby hospital .
eventually , she was able to spend time with her family for 3 months at home .
a - c : mr t1-weighted images with gadolinium show mild enlargement of the left head of the caudate nucleus lesion 3 months after the operation ( a ) , but not presenting recurrence as mass lesions at left frontal and right parietal lesions ( b , c ) .
d - f : spinal mr t1-weighted images with gadolinium revealed a small mass lesion attached to spinal dura mater at the atlas level with no signs of spinal cord compression 3 months after operation .
melanomas are the third most common source of intracranial metastases after breast and lung carcinomas .
the incidence of central nervous system ( cns ) metastases in patients with melanomas ranges from 10% to 40% in clinical studies .
cns metastasis is the most worrisome feature of malignant melanoma , leading directly to death in the majority of patients .
the prognosis for patients with malignant melanoma brain metastasis is poor , with a median survival time ranging between 2 to 10 months .
it is noteworthy that only 10% of patients survive more than 1 year after diagnosis .
an important factor strongly influencing the therapeutic response of patients is the number of metastases .
age , sex , and the time interval from the primary malignant melanoma manifestation to the cerebral metastasis seem not to affect the clinical outcome .
prospective randomized trials have demonstrated the benefit of surgery for the treatment of a single metastasis in the brain .
surgical therapy is ordinarily used for the majority of patients with a single metastatic lesion , but only a few selected patients with multiple metastases in the brain are treated surgically .
recently , konstadoulakis et al . reported that the median survival time of surgical resection for multiple brain metastases was 8 months .
they concluded that performing multiple resections of brain metastases from melanoma is considered a radical approach , especially in young patients with well - controlled systemic disease .
the main aim of a simultaneous one - stage resection for multiple metastatic lesions is limited improvement in the quality of the patient s limited remaining life , so the indications are extremely limited .
the two preconditions are that the patients are young and have well - controlled systemic disease , and that dramatic improvements of neurological symptoms may be expected following the resections .
the surgical options for managing two intracranial lesions are influenced by the presence of another intracranial tumor .
when two lesions are contiguous , they may usually be removed using a single approach , but when they are distant from each other , the surgical approach depends upon the type of lesions .
when one tumor is malignant and the other is benign , the malignant tumor must be removed first .
a second craniotomy may be performed at least 10 to 12 months later , when the benign tumor is large enough and/or symptomatic , or shows a tendency to grow .
when the two lesions are malignant , large , or symptomatic tumors , both lesions may require early resection .
in such cases , there is no consensus as to the best surgical management . in the present case ,
therefore , we selected simultaneous one - stage tumor resection with a left parietal craniotomy and a right frontal craniotomy , and found no adverse events associated with the simultaneous one - stage operation .
the patient s surgical position must be changed under general anesthesia , and the operation time is longer , and more invasive .
nevertheless , the significant reduction of intracranial pressure obtained by surgery may produce immediate and dramatic improvements of the patient s neurological symptoms .
the superiority surgical treatment has over radiotherapy for managing multiple metastatic brain tumors is an immediate reduction in intracranial pressure .
while our patient s survival period was not extended , her symptomatic improvements enabled her to return home and spend time with her family . because it improves the quality of the patient s limited remaining life ,
simultaneous one - stage tumor resection using two approaches is a viable option for the surgical management of multiple metastatic brain tumors , especially in young patients with well - controlled systemic disease .
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