Loss of the Cytoskeletal Protein Pdlim7 Predisposes Mice to Heart Defects and Hemostatic Dysfunction

<div><p>The actin-associated protein Pdlim7 is essential for heart and fin development in zebrafish; however, the expression and function of this PDZ-LIM family member in the mammal has remained unclear. Here, we show that Pdlim7 predominantly localizes to actin-rich structures in mice including the heart, vascular smooth muscle, and platelets. To test the requirement for Pdlim7 in mammalian development and function, we analyzed a mouse strain with global genetic inactivation of Pdlim7. We demonstrate that Pdlim7 loss-of-function leads to significant postnatal mortality. Inactivation of Pdlim7 does not disrupt cardiac development, but causes mild cardiac dysfunction in adult mice. Adult <i>Pdlim7</i>^<i>-/-</i> mice displayed increased mitral and tricuspid valve annulus to body weight ratios. These structural aberrations in <i>Pdlim7</i>^<i>-/-</i> mice were supported by three-dimensional reconstructions of adult cardiac valves, which revealed increased surface area to volume ratios for the mitral and tricuspid valve leaflets. Unexpectedly, we found that loss of Pdlim7 triggers systemic venous and arterial thrombosis, leading to significant mortality shortly after birth in <i>Pdlim7</i>^<i>+/-</i> (11/60) and <i>Pdlim7</i>^<i>-/-</i> (19/35) mice. In line with a prothrombotic phenotype, adult <i>Pdlim7</i>^<i>-/-</i> mice exhibit dramatically decreased tail bleed times compared to controls. These findings reveal a novel and unexpected function for Pdlim7 in maintaining proper hemostasis in neonatal and adult mice. </p> </div

Rational Targeting of Cellular Cholesterol in Diffuse Large B‑Cell Lymphoma (DLBCL) Enabled by Functional Lipoprotein Nanoparticles: A Therapeutic Strategy Dependent on Cell of Origin

Cancer cells have altered metabolism and, in some cases, an increased demand for cholesterol. It is important to identify novel, rational treatments based on biology, and cellular cholesterol metabolism as a potential target for cancer is an innovative approach. Toward this end, we focused on diffuse large B-cell lymphoma (DLBCL) as a model because there is differential cholesterol biosynthesis driven by B-cell receptor (BCR) signaling in germinal center (GC) versus activated B-cell (ABC) DLBCL. To specifically target cellular cholesterol homeostasis, we employed high-density lipoprotein-like nanoparticles (HDL NP) that can generally reduce cellular cholesterol by targeting and blocking cholesterol uptake through the high-affinity HDL receptor, scavenger receptor type B-1 (SCARB1). As we previously reported, GC DLBCL are exquisitely sensitive to HDL NP as monotherapy, while ABC DLBCL are less sensitive. Herein, we report that enhanced BCR signaling and resultant de novo cholesterol synthesis in ABC DLBCL drastically reduces the ability of HDL NPs to reduce cellular cholesterol and induce cell death. Therefore, we combined HDL NP with the BCR signaling inhibitor ibrutinib and the SYK inhibitor R406. By targeting both cellular cholesterol uptake and BCR-associated de novo cholesterol synthesis, we achieved cellular cholesterol reduction and induced apoptosis in otherwise resistant ABC DLBCL cell lines. These results in lymphoma demonstrate that reduction of cellular cholesterol is a powerful mechanism to induce apoptosis. Cells rich in cholesterol require HDL NP therapy to reduce uptake and molecularly targeted agents that inhibit upstream pathways that stimulate de novo cholesterol synthesis, thus, providing a new paradigm for rationally targeting cholesterol metabolism as therapy for cancer

Nitric Oxide-Delivering High-Density Lipoprotein-like Nanoparticles as a Biomimetic Nanotherapy for Vascular Diseases

Disorders of blood vessels cause a range of severe health problems. As a powerful vasodilator and cellular second messenger, nitric oxide (NO) is known to have beneficial vascular functions. However, NO typically has a short half-life and is not specifically targeted. On the other hand, high-density lipoproteins (HDLs) are targeted natural nanoparticles (NPs) that transport cholesterol in the systemic circulation and whose protective effects in vascular homeostasis overlap with those of NO. Evolving the AuNP-templated HDL-like nanoparticles (HDL NPs), a platform of bioinspired HDL, we set up a targeted biomimetic nanotherapy for vascular disease that combines the functions of NO and HDL. A synthetic S-nitrosylated (SNO) phospholipid (1,2-dipalmitoyl-<i>sn</i>-glycero-3-phosphonitrosothioethanol) was synthesized and assembled with S-containing phospholipids and the principal protein of HDL, apolipoprotein A-I, to construct NO-delivering HDL-like particles (SNO HDL NPs). SNO HDL NPs self-assemble under mild conditions similar to natural processes, avoiding the complex postassembly modification needed for most synthetic NO-release nanoparticles. In vitro data demonstrate that the SNO HDL NPs merge the functional properties of NO and HDL into a targeted nanocarrier. Also, SNO HDL NPs were demonstrated to reduce ischemia/reperfusion injury in vivo in a mouse kidney transplant model and atherosclerotic plaque burden in a mouse model of atherosclerosis. Thus, the synthesis of SNO HDL NPs provides not only a bioinspired nanotherapy for vascular disease but also a foundation to construct diversified multifunctional platforms based on HDL NPs in the future

Pathological analysis of non-surviving <i>Pdlim7</i> mutant pups reveals pre-mortem thrombi.

<p><i>Pdlim7</i>^+/- (n=3) and <i>Pdlim7</i>^-/- (n=11) perinatal lethal pups exhibit extensive blood clots in the heart, arteries (arrowhead), and veins (arrows, B-C’) associated with atrial dilation and lung congestion compared to WT (n=7) controls (A-A’). Example of pre-mortem blood clots in the right ventricle (E-E’), umbilical vessel (G-G’), and lung alveoli (I-I’) of a Pdlim7^-/- perinatal lethal pup compared to WT control (D, F, H). Boxes depict location of high magnification image in E’, G’, and I’. Arrows point to attachment of the thrombus to the vessel wall (E’, I’) and arrowhead points to recanalization of the clot (G’). Scale bar = 1mm (A-C’), 50 µm (D-I), 20 µm (G’, I’), and 10µm (E’). </p

Pdlim7 is expressed in platelets and adult Pdlim7 deficient mice display hemostatic dysfunction.

<p>Upon tail clip, blood from 3-week old Pdlim7^-/- (n=9) mice clotted immediately compared to WT (n=16) controls with Pdlim7^+/- mice (n=10) exhibiting an intermediate phenotype (A). Horizontal line denotes mean bleed time for each genotype *p<0.01. Semi-quantitative RT-PCR identifies <i>Pdlim7</i> gene transcripts in WT mouse bone marrow, megakaryocyte cell line Y10 (undifferentiated, day2), and platelets as well as human bone marrow cell line K562 (undifferentiated, 0h; differentiated into proplatelets using TPA for 96h) using GAPDH as a control (B). Western blot analysis of platelets from WT mice demonstrates the presence of Pdlim7 proteins and the absence of Pdlim7 proteins in Pdlim7^-/- mice using GAPDH as a loading control (C). Confocal images of WT platelets spread on glass and immunostained with anti-Pdlim7 antibodies (red) and counterstained for F-actin (green) (D-F). Notably, Pdlim7 is distributed in resting (arrowhead) and spreading platelets (arrow) (E). Scale bar = 5µm.</p

Atrioventricular valves of adult Pdlim7^-/- mice display abnormal morphology.

<p>Histological sections from 3-month old WT and Pdlim7^-/- hearts stained with Masson’s Trichrome (A-D) and Alcian blue (E-F) exhibit normal distribution of collagens (blue, A-D) and glycosaminoglycans (blue, E-F), but elongated mitral valves in Pdlim7^-/- mice (arrows, B, F; n=5) compared to WT controls (A, E; n=3). Scale bar = 100 μm. Representative 3D reconstructions of serial sections of mitral (pink) and tricuspid (turquoise) valves from a WT (G) and Pdlim7^-/- (H) mouse in the same anterodorsolateral perspective. Table providing quantification of increased surface area to volume ratios of atrioventricular valves in Pdlim7^-/- (mitral, n=5; tricuspid, n=4) compared to WT (mitral, n=6; tricuspid, n=5) controls (I). *p<0.04. LV = left ventricle; MV = mitral valve; SA = surface area; TV = tricuspid valve; Vol = volume.</p

Generation of <i>Pdlim7</i> mutant mice.

<p>Schematic representation of the retroviral integration site within intron two of the <i>Pdlim7</i> gene (A). Exons are depicted by yellow boxes with WT and viral-specific genotyping primers represented by arrows and an arrowhead, respectively. Genotyping reveals a 230bp PCR fragment representing the WT allele and a 190bp product for the viral integration (B). Semi-quantitative RT-PCR demonstrates absence of <i>Pdlim7</i> gene transcripts in adult uteri of Pdlim7^-/- mice using GAPDH as control (C). Western blot shows that Pdlim7^-/- and Pdlim7^+/- mice express only -1.18 ± 2.64% and 29.36 ± 14.92% Pdlim7 protein compared to WT controls, respectively (D). Sagittal section through the aorta demonstrates Pdlim7 antibodies (green) localize to filamentous actin (red) of smooth muscle in WT, but not Pdlim7^-/- embryos (E), further verifying loss of Pdlim7 protein in null mice (control DAPI nuclei, blue). LTR = long terminal repeat; SA and SD = splice acceptor and donor sites, respectively; IRES = internal ribosomal entry site; BGEO = lacZ gene; pA = polyadenylation signal; PGK = phosphoglycerate kinase gene; BTK = Bruton’s tyrosine kinase gene. </p

Pdlim7 is dynamically expressed in actin-rich structures throughout murine development.

<p>β-galactosidase staining of whole mount embryos identifies <i>Pdlim7</i> early in the developing heart, forelimbs, hindlimbs, and somites (A-C). In E11.5 and E13.5 heart sections, β-galactosidase staining is localized to the trabeculated regions of the ventricles, atrial walls, and interventricular and atrial septa (D-E); of note: the β-galactosidase reaction time chosen does not reveal the lower levels of Pdlim7 expression in the cardiac cushion or valve cells. Sagittal sections of E18.5 embryos show Pdlim7 expression throughout the smooth muscle, including the lung alveoli, stomach, aorta, and bladder (F). Immunostaining of E11.5 hearts demonstrates Pdlim7 (green) localization to the outflow tract and atrioventricular cushions with MF20 positive myocardial cells (red) (G). Pdlim7 (green) proteins are also detected in the epicardium of E13.5 embryos (arrows, H-I) with MF20 positive myocardial cells (red) and DAPI nuclei (blue). In the aorta, Pdlim7 proteins (green) localize to filamentous actin (red) of smooth muscle, but not PECAM-positive endothelium (white) (arrows, J-L), control DAPI nuclei (blue). Scale bar = 200μm (D-E) and 50 μm (F, I, L). Ao = Aorta; AVC = atrioventricular cushion; Blad = bladder; LA = left atrium; LV = left ventricle; OFT = outflow tract; RA = right atrium; RV = right ventricle. </p

Adult Pdlim7^-/- mice exhibit mild cardiac dysfunction with increased valve annulus dimensions.

<p>Representative Doppler profile in the apical-4 chamber view of mitral valve inflow velocities (A-B) and mitral valve annulus measurement (E-F) from WT (n=8) and Pdlim7^-/- (n=9) mice. Box-and-whisker plots reveal normal fractional shortening (FS) and ejection fraction (EF) in Pdlim7^-/- mice (C), but an increased Tei index (D) and increased mitral and tricuspid annulus dimensions to body weight ratios (G) as compared to controls, *p<0.01. A = late filling; AV = atrioventricular; E = early filling; ET = ejection time; IVCT = isovolumetric contraction time; IVRT = isovolumetric relaxation time; LV = left ventricle; MV = mitral valve; RV = right ventricle.</p