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Decreased human endothelial TSP1. (A) Immunofluorescent staining of TSP1 (red) and collagen IV (green) of human CCM and lesion-free brain tissue. Arrowheads, capillary; arrows, venule; asterisks, vascular lumen of CCM lesions. Bar, <t>100</t> µm ( n = 2). (B and C) HUVECs were transduced with shKrit1 or shControl using lentivirus. (B) KRIT1-depleted HUVECs expressed ∼50% as much TSP1 protein as control cells (SEM, n = 3). (C) KRIT1 shRNA 55% decrease in KRIT1 mRNA as determined by RT-qPCR (SEM, n = 4). **, P
100 Bp Paired Single End, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations"

Article Title: Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20171178

Decreased human endothelial TSP1. (A) Immunofluorescent staining of TSP1 (red) and collagen IV (green) of human CCM and lesion-free brain tissue. Arrowheads, capillary; arrows, venule; asterisks, vascular lumen of CCM lesions. Bar, 100 µm ( n = 2). (B and C) HUVECs were transduced with shKrit1 or shControl using lentivirus. (B) KRIT1-depleted HUVECs expressed ∼50% as much TSP1 protein as control cells (SEM, n = 3). (C) KRIT1 shRNA 55% decrease in KRIT1 mRNA as determined by RT-qPCR (SEM, n = 4). **, P
Figure Legend Snippet: Decreased human endothelial TSP1. (A) Immunofluorescent staining of TSP1 (red) and collagen IV (green) of human CCM and lesion-free brain tissue. Arrowheads, capillary; arrows, venule; asterisks, vascular lumen of CCM lesions. Bar, 100 µm ( n = 2). (B and C) HUVECs were transduced with shKrit1 or shControl using lentivirus. (B) KRIT1-depleted HUVECs expressed ∼50% as much TSP1 protein as control cells (SEM, n = 3). (C) KRIT1 shRNA 55% decrease in KRIT1 mRNA as determined by RT-qPCR (SEM, n = 4). **, P

Techniques Used: Staining, Transduction, shRNA, Quantitative RT-PCR

TSP1 replacement does not suppress the rise in KLF2 and KLF4 after loss of KRIT1. (A and B) Analysis of TSP1, ZO-1, KLF2, and KLF4 mRNA levels by RT-qPCR in freshly isolated microvasculature from mice at P5 and P7 as indicated. Krit1 fl/fl littermate controls, at each developmental stage, were used to calculate percentage increase or decrease in Krit1 ECKO mice using the following formulas: % increase = 100 × ( X − F )/ F and % decrease = 100 × ABS[( F − X )/ F ], where X and F = mRNA abundance in Krit1 ECKO and Krit1 fl/fl BMECs, respectively (SEM, n = 4 or 6). (C) Representative confocal images of retinal vasculature stained for KLF4 (green), TSP1 (red), or isolectin B4 (turquoise). TSP1 is decreased and KLF4 is increased at areas of condensed vasculature ( n = 5 or 6 mice in each group). Bar, 25 µm. (D and E) Analysis of levels of KLF2 and KLF4 mRNA by RT-qPCR from Krit1 ECKO BMEC (D) or cerebellar tissue from Krit1 ECKO mice (E) treated with 3TSR, TSP1, or vehicle compared with Krit1 fl/fl BMEC or Krit1 fl/fl controls. Data are expressed as percentage increase or decrease in Krit1 ECKO using the same formulas as in A and B (SEM, n = 3 or 4 in each group). (F) HUVECs were transduced with lentivirus encoding shKrit1, KLF2, or KLF4, and the increase in KLF2 or KLF4 mRNA relative to cells transduced with lentivirus encoding GFP was measured by RT-qPCR (SEM, n = 4). (G) HUVECs were transduced with lentivirus encoding ShKrit1, KLF2, or KLF4 as described in F, and the decrease of TSP1 mRNA levels was measured relative to cells transduced with EGFP control lentivirus (SEM, n = 4 or 5). (H) Analysis of TSP1 protein levels in HUVECs transduced with lentivirus encoding KLF2 or KLF4 as assessed by Western blot analysis; lentivirus encoding GFP was used as a control (SEM, n = 4). White lines indicate intervening lanes have been spliced out. (I) Loss of endothelial KRIT1 increases expression of KLF2 and KLF4 transcription factors, contributing to CCM formation by downstream effects including suppressed TSP1 expression. 3TSR (TSP1 derivative) reduces CCM lesion formation by replacing functions of TSP1 such as blocking VEGF signaling. Loss of KRIT1 also leads to ROCK activation in a KLF2-dependent manner, and blocking ROCK can also ameliorate CCMs. Thus, blockade of these and other downstream targets of KLF2 and KLF4 may offer a general strategy to reduce CCM formation in humans. *, P
Figure Legend Snippet: TSP1 replacement does not suppress the rise in KLF2 and KLF4 after loss of KRIT1. (A and B) Analysis of TSP1, ZO-1, KLF2, and KLF4 mRNA levels by RT-qPCR in freshly isolated microvasculature from mice at P5 and P7 as indicated. Krit1 fl/fl littermate controls, at each developmental stage, were used to calculate percentage increase or decrease in Krit1 ECKO mice using the following formulas: % increase = 100 × ( X − F )/ F and % decrease = 100 × ABS[( F − X )/ F ], where X and F = mRNA abundance in Krit1 ECKO and Krit1 fl/fl BMECs, respectively (SEM, n = 4 or 6). (C) Representative confocal images of retinal vasculature stained for KLF4 (green), TSP1 (red), or isolectin B4 (turquoise). TSP1 is decreased and KLF4 is increased at areas of condensed vasculature ( n = 5 or 6 mice in each group). Bar, 25 µm. (D and E) Analysis of levels of KLF2 and KLF4 mRNA by RT-qPCR from Krit1 ECKO BMEC (D) or cerebellar tissue from Krit1 ECKO mice (E) treated with 3TSR, TSP1, or vehicle compared with Krit1 fl/fl BMEC or Krit1 fl/fl controls. Data are expressed as percentage increase or decrease in Krit1 ECKO using the same formulas as in A and B (SEM, n = 3 or 4 in each group). (F) HUVECs were transduced with lentivirus encoding shKrit1, KLF2, or KLF4, and the increase in KLF2 or KLF4 mRNA relative to cells transduced with lentivirus encoding GFP was measured by RT-qPCR (SEM, n = 4). (G) HUVECs were transduced with lentivirus encoding ShKrit1, KLF2, or KLF4 as described in F, and the decrease of TSP1 mRNA levels was measured relative to cells transduced with EGFP control lentivirus (SEM, n = 4 or 5). (H) Analysis of TSP1 protein levels in HUVECs transduced with lentivirus encoding KLF2 or KLF4 as assessed by Western blot analysis; lentivirus encoding GFP was used as a control (SEM, n = 4). White lines indicate intervening lanes have been spliced out. (I) Loss of endothelial KRIT1 increases expression of KLF2 and KLF4 transcription factors, contributing to CCM formation by downstream effects including suppressed TSP1 expression. 3TSR (TSP1 derivative) reduces CCM lesion formation by replacing functions of TSP1 such as blocking VEGF signaling. Loss of KRIT1 also leads to ROCK activation in a KLF2-dependent manner, and blocking ROCK can also ameliorate CCMs. Thus, blockade of these and other downstream targets of KLF2 and KLF4 may offer a general strategy to reduce CCM formation in humans. *, P

Techniques Used: Quantitative RT-PCR, Isolation, Mouse Assay, Staining, Transduction, Western Blot, Expressing, Blocking Assay, Activation Assay

TSP1 derivative, 3TSR, prevents CCMs and retinal vascular lesions in Krit1 ECKO mice. (A) Experimental protocol: vehicle or 3TSR (1.6 mg/Kg) was administered by retroorbital plexus injection at P5 and P6, and brains and retinas were analyzed at P7. (B) Prominent lesions are present in the cerebellum of Krit1 ECKO mice, whereas administration of 3TSR suppressed lesion formation. (C) Hematoxylin and eosin staining of cerebellar sections from Krit1 ECKO mice after treatment with 3TSR or vehicle ( n = 4 mice in each group). (D) Representative image of whole-mount P7 retinal vasculature at the angiogenic growth front. The marked area in Krit1 ECKO whole-mount retina shows decreased areas of condensed vascular plexus in Krit1 ECKO treated with 3TSR compared with vehicle-treated Krit1 ECKO littermates (SEM, n = 8 mice in each group). (E) Quantification of lesion coverage in Krit1 ECKO mice treated with 3TSR or Vehicle (SEM, n = 8 mice in each group). (F) Administered 3TSR is present in CCM. 3TSR was injected retroorbitally into a Krit1 ECKO ;Thbs1 −/− mouse. After 30 min, the mouse was killed, and its cerebellar cortex was stained for 3TSR (red, using anti-TSP1 antibodies) and endothelial marker PECAM1 (green); DAPI staining (blue) was used to reveal nuclei. 3TSR is observed in CCM. (G) Higher-magnification images of boxed areas in F. (F and G) Asterisks, vascular lumen of CCM. Bars: (B) 1 mm; (C and F) 100 µm; (D) 200 µm; (G) 50 µm. ***, P
Figure Legend Snippet: TSP1 derivative, 3TSR, prevents CCMs and retinal vascular lesions in Krit1 ECKO mice. (A) Experimental protocol: vehicle or 3TSR (1.6 mg/Kg) was administered by retroorbital plexus injection at P5 and P6, and brains and retinas were analyzed at P7. (B) Prominent lesions are present in the cerebellum of Krit1 ECKO mice, whereas administration of 3TSR suppressed lesion formation. (C) Hematoxylin and eosin staining of cerebellar sections from Krit1 ECKO mice after treatment with 3TSR or vehicle ( n = 4 mice in each group). (D) Representative image of whole-mount P7 retinal vasculature at the angiogenic growth front. The marked area in Krit1 ECKO whole-mount retina shows decreased areas of condensed vascular plexus in Krit1 ECKO treated with 3TSR compared with vehicle-treated Krit1 ECKO littermates (SEM, n = 8 mice in each group). (E) Quantification of lesion coverage in Krit1 ECKO mice treated with 3TSR or Vehicle (SEM, n = 8 mice in each group). (F) Administered 3TSR is present in CCM. 3TSR was injected retroorbitally into a Krit1 ECKO ;Thbs1 −/− mouse. After 30 min, the mouse was killed, and its cerebellar cortex was stained for 3TSR (red, using anti-TSP1 antibodies) and endothelial marker PECAM1 (green); DAPI staining (blue) was used to reveal nuclei. 3TSR is observed in CCM. (G) Higher-magnification images of boxed areas in F. (F and G) Asterisks, vascular lumen of CCM. Bars: (B) 1 mm; (C and F) 100 µm; (D) 200 µm; (G) 50 µm. ***, P

Techniques Used: Mouse Assay, Injection, Staining, Marker

Altered tight junctions are an early phenotypic consequence of Krit1 inactivation. (A) Representative confocal images of ZO-1 (red), claudin5 (CLDN5; turquoise), and VE-cadherin (green) staining in primary BMEC Krit1 ECKO or control Krit1 fl/fl BMECs. Nuclei were counterstained with DAPI (blue; n = 4). Arrows indicate loss of tight junctions but not adherence junctions. (B) Quantification of brain endothelial ZO-1, claudin5, and VE-cadherin protein as assessed by Western blot analysis in Krit1 ECKO compared with Krit1 fl/fl BMEC controls (SEM, n = 3 or 4). (C) Confocal microscopy of cerebellar cortex at P7 stained with anti-PECAM1 (green). (D) Higher-magnification images of boxed areas in C stained for ZO-1 (red), claudin5 (turquoise), and PECAM1 (green). Arrows, staining of tight junction proteins ZO-1 and claudin5; asterisks, vascular lumen of CCM ( n = 3). (E) Quantification of brain endothelial ZO-1, claudin5, and VE-cadherin protein abundance in freshly isolated cerebellar microvasculature in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 3 or 4). (F) Maximum-intensity projection of whole-mount P7 retinal vasculature at the angiogenic growth front stained for ZO-1 (red), claudin5 (turquoise), and an endothelial marker, isolectin B4 (green). (G) Higher-magnification images of boxed areas in F show staining for ZO-1 (red), claudin5 (turquoise), and isolectin B4 (green). (H) Quantification of ZO-1 and claudin5 protein expression in retinal vasculature at the angiogenic front in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 6 mice per group). Bars: (A) 50 µm; (C) 100 µm; (D, F, and G) 25 µm. *, P
Figure Legend Snippet: Altered tight junctions are an early phenotypic consequence of Krit1 inactivation. (A) Representative confocal images of ZO-1 (red), claudin5 (CLDN5; turquoise), and VE-cadherin (green) staining in primary BMEC Krit1 ECKO or control Krit1 fl/fl BMECs. Nuclei were counterstained with DAPI (blue; n = 4). Arrows indicate loss of tight junctions but not adherence junctions. (B) Quantification of brain endothelial ZO-1, claudin5, and VE-cadherin protein as assessed by Western blot analysis in Krit1 ECKO compared with Krit1 fl/fl BMEC controls (SEM, n = 3 or 4). (C) Confocal microscopy of cerebellar cortex at P7 stained with anti-PECAM1 (green). (D) Higher-magnification images of boxed areas in C stained for ZO-1 (red), claudin5 (turquoise), and PECAM1 (green). Arrows, staining of tight junction proteins ZO-1 and claudin5; asterisks, vascular lumen of CCM ( n = 3). (E) Quantification of brain endothelial ZO-1, claudin5, and VE-cadherin protein abundance in freshly isolated cerebellar microvasculature in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 3 or 4). (F) Maximum-intensity projection of whole-mount P7 retinal vasculature at the angiogenic growth front stained for ZO-1 (red), claudin5 (turquoise), and an endothelial marker, isolectin B4 (green). (G) Higher-magnification images of boxed areas in F show staining for ZO-1 (red), claudin5 (turquoise), and isolectin B4 (green). (H) Quantification of ZO-1 and claudin5 protein expression in retinal vasculature at the angiogenic front in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 6 mice per group). Bars: (A) 50 µm; (C) 100 µm; (D, F, and G) 25 µm. *, P

Techniques Used: Staining, Western Blot, Confocal Microscopy, Isolation, Marker, Expressing, Mouse Assay

Loss of KRIT1 inhibits the expression of TSP1. (A) Genome-wide RNA-seq from three independent biological replicates followed by gene ontology analysis of genes differentially expressed in Krit1 ECKO BMECs compared with Krit1 fl/fl BMECs. Each term listed was the top term in a cluster of related terms, and the corrected p-values were calculated according to Benjamini’s method ( Huang et al., 2009 ). (B) Expression levels of differentially expressed genes represented on a scatter plot; fragments per kilobase of transcript per million mapped reads (FPKM) of individual transcripts are represented on a log2 scale. A few of the most highly suppressed and up-regulated genes are labeled. (C) RT-qPCR confirmation of RNA-seq–identified marked decrease in mRNA of extracellular regulators of angiogenesis in Krit1 ECKO BMECs compared with Krit1 fl/fl BMECs (SEM, n = 3). (D) Quantification of TSP1 protein from three independent biological replicates in Krit1 ECKO (KO) and in Krit1 fl/fl (Flox) BMECs (SEM, n = 3). (E) RT-qPCR analysis of isolated brain microvasculature in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 3). (F) Quantification of TSP1 protein from freshly isolated brain microvasculature in Krit1 ECKO (KO) compared with Krit1 fl/fl (Flox) littermate controls (SEM, n = 3). (G) Confocal microscopy of cerebellar cortex stained for TSP1 (red) and endothelial specific marker PECAM1 (green); DAPI staining (blue) was used to reveal nuclei ( n = 3). (H) Higher-magnification images of boxed areas in G. TSP1 protein expression was decreased in CCM from Krit1 ECKO mice (arrows). Asterisks, vascular lumen of CCM lesions. Bars: (G) 100 µm; (H) 25 µm. *, P
Figure Legend Snippet: Loss of KRIT1 inhibits the expression of TSP1. (A) Genome-wide RNA-seq from three independent biological replicates followed by gene ontology analysis of genes differentially expressed in Krit1 ECKO BMECs compared with Krit1 fl/fl BMECs. Each term listed was the top term in a cluster of related terms, and the corrected p-values were calculated according to Benjamini’s method ( Huang et al., 2009 ). (B) Expression levels of differentially expressed genes represented on a scatter plot; fragments per kilobase of transcript per million mapped reads (FPKM) of individual transcripts are represented on a log2 scale. A few of the most highly suppressed and up-regulated genes are labeled. (C) RT-qPCR confirmation of RNA-seq–identified marked decrease in mRNA of extracellular regulators of angiogenesis in Krit1 ECKO BMECs compared with Krit1 fl/fl BMECs (SEM, n = 3). (D) Quantification of TSP1 protein from three independent biological replicates in Krit1 ECKO (KO) and in Krit1 fl/fl (Flox) BMECs (SEM, n = 3). (E) RT-qPCR analysis of isolated brain microvasculature in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 3). (F) Quantification of TSP1 protein from freshly isolated brain microvasculature in Krit1 ECKO (KO) compared with Krit1 fl/fl (Flox) littermate controls (SEM, n = 3). (G) Confocal microscopy of cerebellar cortex stained for TSP1 (red) and endothelial specific marker PECAM1 (green); DAPI staining (blue) was used to reveal nuclei ( n = 3). (H) Higher-magnification images of boxed areas in G. TSP1 protein expression was decreased in CCM from Krit1 ECKO mice (arrows). Asterisks, vascular lumen of CCM lesions. Bars: (G) 100 µm; (H) 25 µm. *, P

Techniques Used: Expressing, Genome Wide, RNA Sequencing Assay, Labeling, Quantitative RT-PCR, Isolation, Confocal Microscopy, Staining, Marker, Mouse Assay

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    Illumina Inc 100 bp paired single end
    Decreased human endothelial TSP1. (A) Immunofluorescent staining of TSP1 (red) and collagen IV (green) of human CCM and lesion-free brain tissue. Arrowheads, capillary; arrows, venule; asterisks, vascular lumen of CCM lesions. Bar, <t>100</t> µm ( n = 2). (B and C) HUVECs were transduced with shKrit1 or shControl using lentivirus. (B) KRIT1-depleted HUVECs expressed ∼50% as much TSP1 protein as control cells (SEM, n = 3). (C) KRIT1 shRNA 55% decrease in KRIT1 mRNA as determined by RT-qPCR (SEM, n = 4). **, P
    100 Bp Paired Single End, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/100 bp paired single end/product/Illumina Inc
    Average 91 stars, based on 1 article reviews
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    Decreased human endothelial TSP1. (A) Immunofluorescent staining of TSP1 (red) and collagen IV (green) of human CCM and lesion-free brain tissue. Arrowheads, capillary; arrows, venule; asterisks, vascular lumen of CCM lesions. Bar, 100 µm ( n = 2). (B and C) HUVECs were transduced with shKrit1 or shControl using lentivirus. (B) KRIT1-depleted HUVECs expressed ∼50% as much TSP1 protein as control cells (SEM, n = 3). (C) KRIT1 shRNA 55% decrease in KRIT1 mRNA as determined by RT-qPCR (SEM, n = 4). **, P

    Journal: The Journal of Experimental Medicine

    Article Title: Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations

    doi: 10.1084/jem.20171178

    Figure Lengend Snippet: Decreased human endothelial TSP1. (A) Immunofluorescent staining of TSP1 (red) and collagen IV (green) of human CCM and lesion-free brain tissue. Arrowheads, capillary; arrows, venule; asterisks, vascular lumen of CCM lesions. Bar, 100 µm ( n = 2). (B and C) HUVECs were transduced with shKrit1 or shControl using lentivirus. (B) KRIT1-depleted HUVECs expressed ∼50% as much TSP1 protein as control cells (SEM, n = 3). (C) KRIT1 shRNA 55% decrease in KRIT1 mRNA as determined by RT-qPCR (SEM, n = 4). **, P

    Article Snippet: RNA libraries were multiplexed and sequenced with 100-bp paired single-end reads (SR100) to a depth of ∼30 million reads per sample on an Illumina HiSeq2500.

    Techniques: Staining, Transduction, shRNA, Quantitative RT-PCR

    TSP1 replacement does not suppress the rise in KLF2 and KLF4 after loss of KRIT1. (A and B) Analysis of TSP1, ZO-1, KLF2, and KLF4 mRNA levels by RT-qPCR in freshly isolated microvasculature from mice at P5 and P7 as indicated. Krit1 fl/fl littermate controls, at each developmental stage, were used to calculate percentage increase or decrease in Krit1 ECKO mice using the following formulas: % increase = 100 × ( X − F )/ F and % decrease = 100 × ABS[( F − X )/ F ], where X and F = mRNA abundance in Krit1 ECKO and Krit1 fl/fl BMECs, respectively (SEM, n = 4 or 6). (C) Representative confocal images of retinal vasculature stained for KLF4 (green), TSP1 (red), or isolectin B4 (turquoise). TSP1 is decreased and KLF4 is increased at areas of condensed vasculature ( n = 5 or 6 mice in each group). Bar, 25 µm. (D and E) Analysis of levels of KLF2 and KLF4 mRNA by RT-qPCR from Krit1 ECKO BMEC (D) or cerebellar tissue from Krit1 ECKO mice (E) treated with 3TSR, TSP1, or vehicle compared with Krit1 fl/fl BMEC or Krit1 fl/fl controls. Data are expressed as percentage increase or decrease in Krit1 ECKO using the same formulas as in A and B (SEM, n = 3 or 4 in each group). (F) HUVECs were transduced with lentivirus encoding shKrit1, KLF2, or KLF4, and the increase in KLF2 or KLF4 mRNA relative to cells transduced with lentivirus encoding GFP was measured by RT-qPCR (SEM, n = 4). (G) HUVECs were transduced with lentivirus encoding ShKrit1, KLF2, or KLF4 as described in F, and the decrease of TSP1 mRNA levels was measured relative to cells transduced with EGFP control lentivirus (SEM, n = 4 or 5). (H) Analysis of TSP1 protein levels in HUVECs transduced with lentivirus encoding KLF2 or KLF4 as assessed by Western blot analysis; lentivirus encoding GFP was used as a control (SEM, n = 4). White lines indicate intervening lanes have been spliced out. (I) Loss of endothelial KRIT1 increases expression of KLF2 and KLF4 transcription factors, contributing to CCM formation by downstream effects including suppressed TSP1 expression. 3TSR (TSP1 derivative) reduces CCM lesion formation by replacing functions of TSP1 such as blocking VEGF signaling. Loss of KRIT1 also leads to ROCK activation in a KLF2-dependent manner, and blocking ROCK can also ameliorate CCMs. Thus, blockade of these and other downstream targets of KLF2 and KLF4 may offer a general strategy to reduce CCM formation in humans. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations

    doi: 10.1084/jem.20171178

    Figure Lengend Snippet: TSP1 replacement does not suppress the rise in KLF2 and KLF4 after loss of KRIT1. (A and B) Analysis of TSP1, ZO-1, KLF2, and KLF4 mRNA levels by RT-qPCR in freshly isolated microvasculature from mice at P5 and P7 as indicated. Krit1 fl/fl littermate controls, at each developmental stage, were used to calculate percentage increase or decrease in Krit1 ECKO mice using the following formulas: % increase = 100 × ( X − F )/ F and % decrease = 100 × ABS[( F − X )/ F ], where X and F = mRNA abundance in Krit1 ECKO and Krit1 fl/fl BMECs, respectively (SEM, n = 4 or 6). (C) Representative confocal images of retinal vasculature stained for KLF4 (green), TSP1 (red), or isolectin B4 (turquoise). TSP1 is decreased and KLF4 is increased at areas of condensed vasculature ( n = 5 or 6 mice in each group). Bar, 25 µm. (D and E) Analysis of levels of KLF2 and KLF4 mRNA by RT-qPCR from Krit1 ECKO BMEC (D) or cerebellar tissue from Krit1 ECKO mice (E) treated with 3TSR, TSP1, or vehicle compared with Krit1 fl/fl BMEC or Krit1 fl/fl controls. Data are expressed as percentage increase or decrease in Krit1 ECKO using the same formulas as in A and B (SEM, n = 3 or 4 in each group). (F) HUVECs were transduced with lentivirus encoding shKrit1, KLF2, or KLF4, and the increase in KLF2 or KLF4 mRNA relative to cells transduced with lentivirus encoding GFP was measured by RT-qPCR (SEM, n = 4). (G) HUVECs were transduced with lentivirus encoding ShKrit1, KLF2, or KLF4 as described in F, and the decrease of TSP1 mRNA levels was measured relative to cells transduced with EGFP control lentivirus (SEM, n = 4 or 5). (H) Analysis of TSP1 protein levels in HUVECs transduced with lentivirus encoding KLF2 or KLF4 as assessed by Western blot analysis; lentivirus encoding GFP was used as a control (SEM, n = 4). White lines indicate intervening lanes have been spliced out. (I) Loss of endothelial KRIT1 increases expression of KLF2 and KLF4 transcription factors, contributing to CCM formation by downstream effects including suppressed TSP1 expression. 3TSR (TSP1 derivative) reduces CCM lesion formation by replacing functions of TSP1 such as blocking VEGF signaling. Loss of KRIT1 also leads to ROCK activation in a KLF2-dependent manner, and blocking ROCK can also ameliorate CCMs. Thus, blockade of these and other downstream targets of KLF2 and KLF4 may offer a general strategy to reduce CCM formation in humans. *, P

    Article Snippet: RNA libraries were multiplexed and sequenced with 100-bp paired single-end reads (SR100) to a depth of ∼30 million reads per sample on an Illumina HiSeq2500.

    Techniques: Quantitative RT-PCR, Isolation, Mouse Assay, Staining, Transduction, Western Blot, Expressing, Blocking Assay, Activation Assay

    TSP1 derivative, 3TSR, prevents CCMs and retinal vascular lesions in Krit1 ECKO mice. (A) Experimental protocol: vehicle or 3TSR (1.6 mg/Kg) was administered by retroorbital plexus injection at P5 and P6, and brains and retinas were analyzed at P7. (B) Prominent lesions are present in the cerebellum of Krit1 ECKO mice, whereas administration of 3TSR suppressed lesion formation. (C) Hematoxylin and eosin staining of cerebellar sections from Krit1 ECKO mice after treatment with 3TSR or vehicle ( n = 4 mice in each group). (D) Representative image of whole-mount P7 retinal vasculature at the angiogenic growth front. The marked area in Krit1 ECKO whole-mount retina shows decreased areas of condensed vascular plexus in Krit1 ECKO treated with 3TSR compared with vehicle-treated Krit1 ECKO littermates (SEM, n = 8 mice in each group). (E) Quantification of lesion coverage in Krit1 ECKO mice treated with 3TSR or Vehicle (SEM, n = 8 mice in each group). (F) Administered 3TSR is present in CCM. 3TSR was injected retroorbitally into a Krit1 ECKO ;Thbs1 −/− mouse. After 30 min, the mouse was killed, and its cerebellar cortex was stained for 3TSR (red, using anti-TSP1 antibodies) and endothelial marker PECAM1 (green); DAPI staining (blue) was used to reveal nuclei. 3TSR is observed in CCM. (G) Higher-magnification images of boxed areas in F. (F and G) Asterisks, vascular lumen of CCM. Bars: (B) 1 mm; (C and F) 100 µm; (D) 200 µm; (G) 50 µm. ***, P

    Journal: The Journal of Experimental Medicine

    Article Title: Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations

    doi: 10.1084/jem.20171178

    Figure Lengend Snippet: TSP1 derivative, 3TSR, prevents CCMs and retinal vascular lesions in Krit1 ECKO mice. (A) Experimental protocol: vehicle or 3TSR (1.6 mg/Kg) was administered by retroorbital plexus injection at P5 and P6, and brains and retinas were analyzed at P7. (B) Prominent lesions are present in the cerebellum of Krit1 ECKO mice, whereas administration of 3TSR suppressed lesion formation. (C) Hematoxylin and eosin staining of cerebellar sections from Krit1 ECKO mice after treatment with 3TSR or vehicle ( n = 4 mice in each group). (D) Representative image of whole-mount P7 retinal vasculature at the angiogenic growth front. The marked area in Krit1 ECKO whole-mount retina shows decreased areas of condensed vascular plexus in Krit1 ECKO treated with 3TSR compared with vehicle-treated Krit1 ECKO littermates (SEM, n = 8 mice in each group). (E) Quantification of lesion coverage in Krit1 ECKO mice treated with 3TSR or Vehicle (SEM, n = 8 mice in each group). (F) Administered 3TSR is present in CCM. 3TSR was injected retroorbitally into a Krit1 ECKO ;Thbs1 −/− mouse. After 30 min, the mouse was killed, and its cerebellar cortex was stained for 3TSR (red, using anti-TSP1 antibodies) and endothelial marker PECAM1 (green); DAPI staining (blue) was used to reveal nuclei. 3TSR is observed in CCM. (G) Higher-magnification images of boxed areas in F. (F and G) Asterisks, vascular lumen of CCM. Bars: (B) 1 mm; (C and F) 100 µm; (D) 200 µm; (G) 50 µm. ***, P

    Article Snippet: RNA libraries were multiplexed and sequenced with 100-bp paired single-end reads (SR100) to a depth of ∼30 million reads per sample on an Illumina HiSeq2500.

    Techniques: Mouse Assay, Injection, Staining, Marker

    Altered tight junctions are an early phenotypic consequence of Krit1 inactivation. (A) Representative confocal images of ZO-1 (red), claudin5 (CLDN5; turquoise), and VE-cadherin (green) staining in primary BMEC Krit1 ECKO or control Krit1 fl/fl BMECs. Nuclei were counterstained with DAPI (blue; n = 4). Arrows indicate loss of tight junctions but not adherence junctions. (B) Quantification of brain endothelial ZO-1, claudin5, and VE-cadherin protein as assessed by Western blot analysis in Krit1 ECKO compared with Krit1 fl/fl BMEC controls (SEM, n = 3 or 4). (C) Confocal microscopy of cerebellar cortex at P7 stained with anti-PECAM1 (green). (D) Higher-magnification images of boxed areas in C stained for ZO-1 (red), claudin5 (turquoise), and PECAM1 (green). Arrows, staining of tight junction proteins ZO-1 and claudin5; asterisks, vascular lumen of CCM ( n = 3). (E) Quantification of brain endothelial ZO-1, claudin5, and VE-cadherin protein abundance in freshly isolated cerebellar microvasculature in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 3 or 4). (F) Maximum-intensity projection of whole-mount P7 retinal vasculature at the angiogenic growth front stained for ZO-1 (red), claudin5 (turquoise), and an endothelial marker, isolectin B4 (green). (G) Higher-magnification images of boxed areas in F show staining for ZO-1 (red), claudin5 (turquoise), and isolectin B4 (green). (H) Quantification of ZO-1 and claudin5 protein expression in retinal vasculature at the angiogenic front in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 6 mice per group). Bars: (A) 50 µm; (C) 100 µm; (D, F, and G) 25 µm. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations

    doi: 10.1084/jem.20171178

    Figure Lengend Snippet: Altered tight junctions are an early phenotypic consequence of Krit1 inactivation. (A) Representative confocal images of ZO-1 (red), claudin5 (CLDN5; turquoise), and VE-cadherin (green) staining in primary BMEC Krit1 ECKO or control Krit1 fl/fl BMECs. Nuclei were counterstained with DAPI (blue; n = 4). Arrows indicate loss of tight junctions but not adherence junctions. (B) Quantification of brain endothelial ZO-1, claudin5, and VE-cadherin protein as assessed by Western blot analysis in Krit1 ECKO compared with Krit1 fl/fl BMEC controls (SEM, n = 3 or 4). (C) Confocal microscopy of cerebellar cortex at P7 stained with anti-PECAM1 (green). (D) Higher-magnification images of boxed areas in C stained for ZO-1 (red), claudin5 (turquoise), and PECAM1 (green). Arrows, staining of tight junction proteins ZO-1 and claudin5; asterisks, vascular lumen of CCM ( n = 3). (E) Quantification of brain endothelial ZO-1, claudin5, and VE-cadherin protein abundance in freshly isolated cerebellar microvasculature in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 3 or 4). (F) Maximum-intensity projection of whole-mount P7 retinal vasculature at the angiogenic growth front stained for ZO-1 (red), claudin5 (turquoise), and an endothelial marker, isolectin B4 (green). (G) Higher-magnification images of boxed areas in F show staining for ZO-1 (red), claudin5 (turquoise), and isolectin B4 (green). (H) Quantification of ZO-1 and claudin5 protein expression in retinal vasculature at the angiogenic front in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 6 mice per group). Bars: (A) 50 µm; (C) 100 µm; (D, F, and G) 25 µm. *, P

    Article Snippet: RNA libraries were multiplexed and sequenced with 100-bp paired single-end reads (SR100) to a depth of ∼30 million reads per sample on an Illumina HiSeq2500.

    Techniques: Staining, Western Blot, Confocal Microscopy, Isolation, Marker, Expressing, Mouse Assay

    Loss of KRIT1 inhibits the expression of TSP1. (A) Genome-wide RNA-seq from three independent biological replicates followed by gene ontology analysis of genes differentially expressed in Krit1 ECKO BMECs compared with Krit1 fl/fl BMECs. Each term listed was the top term in a cluster of related terms, and the corrected p-values were calculated according to Benjamini’s method ( Huang et al., 2009 ). (B) Expression levels of differentially expressed genes represented on a scatter plot; fragments per kilobase of transcript per million mapped reads (FPKM) of individual transcripts are represented on a log2 scale. A few of the most highly suppressed and up-regulated genes are labeled. (C) RT-qPCR confirmation of RNA-seq–identified marked decrease in mRNA of extracellular regulators of angiogenesis in Krit1 ECKO BMECs compared with Krit1 fl/fl BMECs (SEM, n = 3). (D) Quantification of TSP1 protein from three independent biological replicates in Krit1 ECKO (KO) and in Krit1 fl/fl (Flox) BMECs (SEM, n = 3). (E) RT-qPCR analysis of isolated brain microvasculature in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 3). (F) Quantification of TSP1 protein from freshly isolated brain microvasculature in Krit1 ECKO (KO) compared with Krit1 fl/fl (Flox) littermate controls (SEM, n = 3). (G) Confocal microscopy of cerebellar cortex stained for TSP1 (red) and endothelial specific marker PECAM1 (green); DAPI staining (blue) was used to reveal nuclei ( n = 3). (H) Higher-magnification images of boxed areas in G. TSP1 protein expression was decreased in CCM from Krit1 ECKO mice (arrows). Asterisks, vascular lumen of CCM lesions. Bars: (G) 100 µm; (H) 25 µm. *, P

    Journal: The Journal of Experimental Medicine

    Article Title: Thrombospondin1 (TSP1) replacement prevents cerebral cavernous malformations

    doi: 10.1084/jem.20171178

    Figure Lengend Snippet: Loss of KRIT1 inhibits the expression of TSP1. (A) Genome-wide RNA-seq from three independent biological replicates followed by gene ontology analysis of genes differentially expressed in Krit1 ECKO BMECs compared with Krit1 fl/fl BMECs. Each term listed was the top term in a cluster of related terms, and the corrected p-values were calculated according to Benjamini’s method ( Huang et al., 2009 ). (B) Expression levels of differentially expressed genes represented on a scatter plot; fragments per kilobase of transcript per million mapped reads (FPKM) of individual transcripts are represented on a log2 scale. A few of the most highly suppressed and up-regulated genes are labeled. (C) RT-qPCR confirmation of RNA-seq–identified marked decrease in mRNA of extracellular regulators of angiogenesis in Krit1 ECKO BMECs compared with Krit1 fl/fl BMECs (SEM, n = 3). (D) Quantification of TSP1 protein from three independent biological replicates in Krit1 ECKO (KO) and in Krit1 fl/fl (Flox) BMECs (SEM, n = 3). (E) RT-qPCR analysis of isolated brain microvasculature in Krit1 ECKO compared with Krit1 fl/fl littermate controls (SEM, n = 3). (F) Quantification of TSP1 protein from freshly isolated brain microvasculature in Krit1 ECKO (KO) compared with Krit1 fl/fl (Flox) littermate controls (SEM, n = 3). (G) Confocal microscopy of cerebellar cortex stained for TSP1 (red) and endothelial specific marker PECAM1 (green); DAPI staining (blue) was used to reveal nuclei ( n = 3). (H) Higher-magnification images of boxed areas in G. TSP1 protein expression was decreased in CCM from Krit1 ECKO mice (arrows). Asterisks, vascular lumen of CCM lesions. Bars: (G) 100 µm; (H) 25 µm. *, P

    Article Snippet: RNA libraries were multiplexed and sequenced with 100-bp paired single-end reads (SR100) to a depth of ∼30 million reads per sample on an Illumina HiSeq2500.

    Techniques: Expressing, Genome Wide, RNA Sequencing Assay, Labeling, Quantitative RT-PCR, Isolation, Confocal Microscopy, Staining, Marker, Mouse Assay