Table 2.

Summary of gene expression in Groups 4.1–4.8.

Description of comparisons, normalization	Coating	Donors	Treatment	Marker of inflammation	Markers of osteogenic differentiation			Markers of myofibroblastic differentiation		Statistical analysis
				ICAM1	BMP2	RUNX2	POSTN	ACTA2	CNN1
Control vs. Treatment (LPS, Flex or both) Mean of RQ values of control group (Flex-LPS-) = 1	Collagen	Healthy (n = 6)	Flex-LPS+	30.1 ± 22.9 p = 0.03	6 ± 1.6 p = 0.9	3.3 ± 3.4 p = 0.9	0.8 ± 0.5 p = 0.99	0.4 ± 0.4 p = 0.03	0.5 ± 0.2 p = 0.001	One-way ANOVA with Dunnett's multiple comparison post-test (for parametric data) or non-parametric test (Kruskal-Wallis test) with Dunn's multiple comparisons post-test (for non-parametric data)
			Flex+LPS-	4.8 ± 5 p = 0.97	1.5 ± 0.7 p > 0.99	2.7 ± 2.2 p = 0.9	1.8 ± 1.7 p = 0.99	0.3 ± 0.3 p = 0.003	0.6 ± 0.3 p = 0.005
			Flex+LPS+	30 ± 27.3 p = 0.03	33.9 ± 35.1 p = 0.01	11.6 ± 12.4 p = 0.03	1.1 ± 0.4 p = 0.5	0.3 ± 0.4 p = 0.009	0.4 ± 0.2 p < 0.0001
		Calcified (n = 6)	Flex-LPS+	41.9 ± 30.2 p = 0.004	5.1 ± 3.4 p = 0.02	2.3 ± 2.3 p = 0.99	0.3 ± 0.1 p = 0.001	0.5 ± 0.2 p = 0.0006	0.2 ± 0.1 p < 0.0001
			Flex+LPS-	11.1 ± 9.8 p = 0.7	2.9 ± 1.7 p = 0.4	0.4 ± 0.3 p = 0.99	0.6 ± 0.2 p = 0.2	0.9 ± 0.2 p = 0.3	1 ± 0.3 p = 0.98
			Flex+LPS+	31.2 ± 20.4 p = 0.03	7 ± 3 p = 0.0009	17.5 ± 19.5 p = 0.03	0.5 ± 0.4 p = 0.06	0.5 ± 0.2 p = 0.001	0.4 ± 0.2 p = 0.0002
	Elastin	Healthy (n = 6)	Flex-LPS+	18.7 ± 23.7 p = 0.2	10.8 ± 14.2 p = 0.3	1.3 ± 1.2 p = 0.99	1.4 ± 1.5 p > 0.99	0.8 ± 0.6 p = 0.7	0.8 ± 0.3 p > 0.99
			Flex+LPS-	0.9 ± 0.4 p > 0.99	7.2 ± 8 p = 0.2	4.9 ± 4.5 p = 0.08	2.1 ± 2.6 p > 0.99	c0.5 ± 0.2 p = 0.03	0.7 ± 0.5 p = 0.2
			Flex+LPS+	23.6 ± 19.1 p = 0.54	17.6 ± 13.8 p = 0.003	4.2 ± 3.5 p = 0.2	1.6 ± 1.7 p > 0.99	0.4 ± 0.3 p = 0.008	0.5 ± 0.6 p = 0.06
		Calcified (n = 6)	Flex-LPS+	5.4 ± 5.4 p = 0.98	0.7 ± 0.4 p = 0.99	0.4 ± 0.6 p = 0.9	0.8 ± 0.6 p = 0.5	0.9 ± 0.7 p = 0.9	0.7 ± 0.3 p = 0.1
			Flex+LPS-	33.6 ± 35.8 p = 0.1	13.4 ± 9.6 p = 0.03	1.4 ± 1.4 p = 0.97	0.7 ± 0.7 p = 0.2	0.3 ± 0.1 p = 0.03	0.5 ± 0.3 p = 0.003
			Flex+LPS+	39.5 ± 41.9 p = 0.07	17.1 ± 12.2 p = 0.05	3.5 ± 3.6 p = 0.1	1 ± 0.7 p > 0.99	0.4 ± 0.3 p = 0.04	0.3 ± 0.2 p < 0.0001
Collagen vs. Elastin Mean of RQ values of every treatment group of cells cultured on collagen = 1	Elastin/ Collagen	Healthy (n = 6)	Flex-LPS+	0.6 ± 0.9 p = 0.51	1.8 ± 2.4 p > 0.9	0.4 ± 0.3 p = 0.3	1.7 ± 1.8 p = 0.8	2.1 ± 1.5 p = 0.03	1.5 ± 0.6 p = 0.03	Paired t-test (for parametric data) or Wilcoxon matched-pairs signed rank test (for non-parametric data)
			Flex+LPS-	0.7 ± 1.3 p > 0.99	4.7 ± 5.2 p = 0.09	1.8 ± 1.7 p = 0.3	1.2 ± 1.5 p = 0.7	1.8 ± 0.9 p = 0.06	1.1 ± 0.9 p > 0.99
			Flex+LPS+	0.9 ± 0.6 p = 0.7	0.5 ± 0.4 p = 0.4	0.4 ± 0.3 p = 0.1	1.6 ± 1.8 p = 0.6	1.1 ± 0.8 p = 0.9	1.5 ± 1.6 p > 0.99
		Calcified (n = 6)	Flex-LPS+	0.1 ± 0.1 p = 0.02	0.2 ± 0.1 p = 0.03	0.1 ± 0.2 p = 0.03	3.2 ± 2.4 p = 0.03	1.8 ± 1.5 p = 0.03	3.2 ± 1.6 p = 0.008
							Flex+LPS-	3 ± 3.2 p = 0.2	4.7 ± 3.4 p = 0.04	3.7 ± 3.5 p = 0.2	1.1 ± 1.3 p > 0.9	0.4 ± 0.2 p = 0.03	0.5 ± 0.3 p = 0.006
			Flex+LPS+	1.1 ± 1.2 p = 0.9	2.5 ± 1.8 p = 0.07	0.2 ± 0.2 p = 0.06	1.9 ± 1.3 p = 0.3	0.7 ± 0.5 p = 0.1	0.6 ± 0.4 p = 0.08
Healthy vs. Calcified Mean of RQ values of every treatment group of cells from healthy valves = 1	Collagen	Calcified/Healthy (n = 6)	Flex-LPS+	1.4 ± 1 p = 0.5	0.8 ± 0.6 p = 0.5	0.9 ± 0.6 p = 0.8	0.3 ± 0.2 p = 0.03	1.3 ± 0.6 p = 0.4	0.4 ± 0.2 p = 0.01	Unpaired t-test (for parametric data) or Mann-Whitney test (for non-parametric data)
			Flex+LPS-	2.3 ± 2.1 p = 0.2	1.9 ± 1.1 p = 0.1	0.1 ± 0.09 p = 0.05	0.3 ± 0.1 p = 0.1	2.2 ± 0.4 p = 0.09	1.7 ± 0.6 p = 0.05
			Flex+LPS+	1 ± 0.7 p > 0.99	0.2 ± 0.1 p = 0.09	1.5 ± 1.7 p = 0.5	0.5 ± 0.4 p = 0.07	1.5 ± 0.5 p = 0.2	1.2 ± 0.5 p = 0.5
	Elastin		Flex-LPS+	0.3 ± 0.3 p = 0.2	0.1 ± 0.04 p = 0.1	0.3 ± 0.4 p = 0.2	0.6 ± 0.4 p = 0.8	1.1 ± 0.9 p = 0.9	0.9 ± 0.4 p = 0.7
			Flex+LPS-	10 ± 10.6 p = 0.09	1.9 ± 1.3 p = 0.2	0.3 ± 0.3 p = 0.1	0.3 ± 0.4 p = 0.3	0.7 ± 0.3 p = 0.3	0.8 ± 0.4 p = 0.7
			Flex+LPS+	1.7 ± 1.8 p = 0.4	1 ± 0.7 p = 0.9	0.8 ± 0.8 p = 0.8	0.6 ± 0.4 p = 0.6	1.1 ± 0.8 p = 0.9	0.5 ± 0.3 p = 0.7

Relative gene expression of markers related to inflammation, osteogenic and myofibroblastic differentiation in valve interstitial cells isolated from healthy (n = 6) and calcified (n = 6) valves, cultured on collagen or elastin coatings and stimulated by LPS and stretch (“Flex”), or both. Data are normalized depending on the groups that are compared and presented as mean ± SD. Upregulated genes are highlighted in green, downregulated genes are highlighted in red. p-values < 0.05 were considered statistically significant.