Matrix metalloproteinases -8 and -9 in the Airways, Blood and Urine During Exacerbations of COPD

Figures & data

Figure 1.  Consort diagram and study protocol for the COPD exacerbation cohort. Other withdrawal includes inability to complete protocol due to illness, further disease exacerbation, inability to attend visit due to co-morbidity or frailty and withdrawal of consent.

Table 1.  Characteristics of patients recruited to the study, attending follow-up visit (F/U) and confirmed to have COPD

	All patients Mean (S.D.)	Did not attend final visit Mean (S.D.)	Attended final visit Mean (S.D.)	Final COPD cohort Mean (S.D.)
Demographic data
n	153	64	89	72
Age (years)	69 (10)	71 (9)	68 (10)	68 (9)
Gender M/F (n)	101/52	40/24	61/28	49/23
Current smoker (n)	62	24	38	33
Pack-years	47 (26)	43 (23)	50 (27)	51 (25)
Disease severity
FEV1 (litres)	1.25 (0.54)		1.25 (0.54)	1.19 (0.49)
FEV1 (% predicted)	48.72 (19.30)		48.72 (19.30)	46.25 (16.13)
FEV1/FVC	0.49 (0.14)		0.49 (0.14)	0.46 (0.11)
GOLD stage (n)
Unknown or NA	77		17	0
1	3		2	2
2	26		26	26
3	35		35	35
4	9		9	9
Admission CAT score	29.84 (6.44)	32.29 (6.22)	28.63 (6.36)	28.03 (6.66)
Follow-up CAT score	21.33 (7.74)		21.33 (7.74)	20.78 (7.79)
MRC grade	3.95 (1.02)	4.13 (1.02	3.82 (1.02)	3.69 (1.02)
Outcome
Duration of stay (days)	5.17 (6.08)	6.5 (7.70)	5.21 (4.54)	4.86 (3.94)
Death	5	5	0	0

Mean values for age, gender, MRC score, CAT score were not significantly different between those completing and not completing the study.

NB: GOLD scores are only attributed to those with confirmed COPD.

Figure 2.  MMP expression is different in sputum, serum and urine. (a) Representative gelatin zymogram of serum, sputum and urine of a single patient showing different MMP species and complexes in separate compartments. Visit 1: admission during exacerbation, visit 2: exacerbation days 5–7, visit 3: recovery days 28–30. NGAL, neutrophil associated lipocalin. (b) Quantitation of values of active MMP-9 in sputum, serum and urine from 15 patients. (c) Relative change in MMP-8, -9 and active MMP-9 in serum and urine from exacerbation day 1 to recovery days 28–30 in 53 patients. The direction of change was not consistent for any of the MMP species tested.

Table 2.  Comparison of MMP measures within sputum, serum and urine obtained at the same visit

	n	MMP-8	MMP-9	Active MMP-9
Sputum vs. serum	15	not tested	not tested	p>0.999, r=0.000
Sputum vs. urine	11	not tested	not tested	p=0.155, r=0.464
Serum vs. urine	53	p=.205, r=-.086	p=.607, r=.035	p=0.281, r=0.073

Table 3.  Association between MMP values and lung function in 72 patients at recovery visit

		FEV1	FEV1/FVC
Serum	MMP-8^*	p=0.625. r=-0.059	p=0.977, r=-0.003
	MMP-9^*	p=0.564, r=-0.070	p=0.657, r=-0.054
	Active MMP-9^†	p=0.392, r=0.103	p=0.797, r=-0.031
Urine	MMP-8^**	p=0.337, r=-0.122	p=0.556, r=-0.075
	MMP-9^**	p=0.145, r=-0.184	p=0.242, r=-0.148
	Active MMP-9^†	p=0.769, r=-0.038	p=0.194, r=-0.164

*ng/ml, ^†arbitrary units, ^**ng/mmol creatinine.

Table 4. Median (IQR) MMP levels in COPD patient serum and urine measured during exacerbations and recovery

		Exacerbation day 1	Exacerbation day 5–7	Recovery day 28–30
Serum	Total MMP-9^*	438.6 (262.7–633.0)	462.4 (267.9–742.0)	382 (218–557.6)
	Active MMP-9^†	0.073 (0.046–0.073)	0.067 (0.049–0.11)	0.081 (0.049–0.12)
	Total MMP-8^*	15.1 (2.1–38.6)	14.8 (0.001–33.0)	12.0 (1.1–29.5)
Urine	Total MMP-9^**	181.2 (0.002–509.1)	198.8 (0.002–525.0)	285.7 (1.95–757.5)
	Active MMP-9^†	0.001 (0.001–0.003)	0.002 (0.001–0.004)	0.002 (0.001–0.006)
	Total MMP-8^**	108.9 (7.3–441.4)	114 (0.004–517.2)	85.9 (0.004–556.6)

*ng/ml, ^†arbitrary units, ^**ng/mmol creatinine.

Supplemental Table 1 . Characteristics of stable patients measured 1 week apart

	Visit 1	Visit 2	P value
Gender (M/F)	13/9
Age	68.4 (5.34)
Smoker (current/ex)	3/22
MRC grade	2.81 (1.03)	2.82 (1.10)	>0.99
CAT score	18.7 (7.50)	18.7 (9.22)	>0.99
% predicted FEV1	57.6 (17.9)	57.1 (18.65)	0.55
FEV1/FVC	56.2 (12.1)	54.1 (13.5)	0.095

Supplemental Table 2 . MMPs values at two time points in stable patient with COPD

		Visit 1	Visit 2	P value
Serum	Total MMP-8^*	16.47 (8.54–34.5)	18.33 (11.76–39.14)	0.57
	Total MMP-9^*	437.4 (232.9–590.5)	431.9 (352.6–638.7)	0.46
	Active MMP-9^†	0.049 (0.036–0.072)	0.055 (0.035–0.074)	0.38
	Active/total MMP-9^†	0.227 (0.143–0.255)	0.208 (0.134–0.250)	0.59
Urine	Total MMP-8^*	0.004 (0.004–102.0)	53.5 (0.004–448.2)	0.003
	Total MMP-9^*	261.4 (0.002–401.3)	347.5 (132.4–1743)	0.24
	Active MMP-9^†	0.001 (0.0007–0.003)	0.001 (0.0004–0.002)	0.61
	Active/total MMP-9^†	0.476 (0.220–0.512)	0.427 (0.260–0.523)	0.44

Median (IQR) serum and urine levels of MMPs in stable patients with COPD 1 week apart. ^*ng/ml, ^†arbitrary units, ^**ng/mmol creatinine.

Supplemental Table 3 . Baseline serum and urine levels of MMPs compared to FEV₁ and FEV₁/FVC

	Sample	FEV1	FEV1/FVC
Serum	MMP-8^*	p=0.625. r=–0.059	p=0.977, r=–0.003
	MMP-9^*	p=0.564, r=–0.070	p=0.657, r=–0.054
	Active MMP-9^†	p=0.392, r=0.103	p=0.797, r=–0.031
Urine	MMP-8^**	p=0.337, r=–0.122	p=0.556, r=–0.075
	MMP-9^**	p=0.145, r=–0.184	p=0.242, r=–0.148
	Active MMP-9^†	p=0.769, r=–0.0375	p=0.194, r=–0.164

^*ng/ml, ^†arbitrary units, ^**ng/mmol creatinine.

Figure 3.  MMP-8 and -9 are not associated with severity of COPD. Graphs show individual MMP levels in serum and urine at different GOLD stages. (a and b) Serum MMP-8 and -9, (c and d) urine MMP-8 and -9, (e) serum MMP-9 and CAT score, (f) serum MMP-9 and MRC dyspnoea score.

Figure 4.  Change in MMPs between exacerbations and convalescence in individual patients. (a-c) Serum MMP-9, -8 and active MMP-9, respectively, (d-f) Urine MMP-9, -8 and active MMP-9, respectively.

Supplemental Table 4 . MMP levels in serum and urine at admission compared with length of hospital stay

		n	LOS ≤7 days	n	LOS >7 days or death	P value
Serum	MMP-8^*	56	14.43 (2.139–38.58)	19	16.03 (2.60-40.42)	0.64
	MMP-9^*		409.3 (248.4–562.8)		477.3 (299.1-805.4)	0.12
	Active MMP-9^†		0.072 (0.047–0.099)		0.074 (0.046-0.122)	0.82
Urine	MMP-8^**	49	177.3 (0.004–446.8)	18	111 (25.42-608.3)	0.94
	MMP-9^**		155.1 (0.002–512.8)		355.7 (60.19-788.5)	0.23
	Active MMP-9^†		0.0015 (0.0009–0.002)		0.0014 (0.0007-0.003)	0.84

*ng/ml, ^†arbitrary units, ^**ng/mmol creatinine. Median (IQR).

LOS: length of hospital stay.

Figure 5.  Serum MMP-8 and -9 are correlated with serum neutrophils. (a) serum MMP-9, (b) serum MMP-8. Neutrophil count × 10⁶ ml^-1.

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