Pulmonary hypertension : the role and place of PDGF

Pulmonary hypertension is a severe condition, leading to right heart dysfunction and preterm death. For pulmonary arterial hypertension (PAH), a disease group in which the primary pathology resides within the pulmonary pre-capillary vessels, several specific therapies are in clinical use, but unfortunately the prognosis is still grim. Available PAH therapy mainly targets vasoconstriction and the progressive vascular remodeling is not adequately suppressed. Considering biological similarities to malignancies, hypotheses and therapies from cancer research have been tested in PAH. An example of this is imatinib, originally designed to target a mutated receptor in chronic myeloid leukemia, and found to also inhibit platelet-derived growth factor (PDGF) signaling. Albeit to some extent efficient, imatinib is unspecific and leads to severe side effects in PAH patients. Previous studies have found PDGF receptor b and its ligand, PDGF-B, to be implicated in PAH. PDGF signaling is known to induce cell proliferation, migration, and extracellular matrix deposition. Additionally, PDGF-B contains a retention motif that binds to matrix proteoglycans, such as perlecan. Perlecan, which has previously been shown to affect vascular remodeling in the systemic circulation, has here been investigated in the pulmonary circulation. Further, the role of PDGF-D, the other known ligand of PDGF receptor b, has in this thesis been characterized in physiology as well as in pulmonary hypertension. Paper I and II combined describes how pulmonary vascular remodeling could be altered by either targeting the PDGF-B retention motif or perlecan heparan sulfate (HS). In development, perivascular smooth muscle cells and pericytes propagate towards an extracellular PDGF-B gradient. Our findings support previous reports on similar mechanisms also in hypoxia-induced pulmonary vascular remodeling. Further, we show that perlecan HS promotes fibroblast growth factor signaling, another important mitogen for smooth muscle cells and pericytes. In paper III, effects of PDGF-D deletion were thoroughly characterized. Pdgfd-/- mice were shown to be viable and healthy, however a mild cardiovascular phenotype, including discrete alterations in pericyte attachment to cardiac microvessels, was found. In Paper IV the role of PDGF-D in PH was explored. It was shown to be present in vascular lesions of PAH patients and recombinant PDGF-D potently induced proliferation of human and mouse pulmonary arterial smooth muscle cells in vitro. This suggested that PDGF-D could be a driver of pulmonary vascular remodeling. However, Pdgfd-/- mice were not protected against disease and hence, PDGF-D seems to be a redundant mitogen in hypoxia-induced PH. The collected work of this thesis highlights the importance of spatial distribution of growth factors and prompts future PAH studies to take the extracellular matrix into consideration

Mice Lacking Platelet-Derived Growth Factor D Display a Mild Vascular Phenotype

Platelet-derived growth factor D (PDGF-D) is the most recently discovered member of the PDGF family. PDGF-D signals through PDGF receptor beta, but its biological role remains largely unknown. In contrast to other members of the PDGF family of growth factors, which have been extensively investigated using different knockout approaches in mice, PDGF-D has until now not been characterized by gene inactivation in mice. Here, we present the phenotype of a constitutive Pdgfd knockout mouse model (Pdgfd(-/-)), carrying a LacZ reporter used to visualize Pdgfd promoter activity. Inactivation of the Pdgfd gene resulted in a mild phenotype in C57BL/6 mice, and the offspring was viable, fertile and generally in good health. We show that Pdgfd reporter gene activity was consistently localized to vascular structures in both postnatal and adult tissues. The expression was predominantly arterial, often localizing to vascular bifurcations. Endothelial cells appeared to be the dominating source for Pdgfd, but reporter gene activity was occasionally also found in sub-populations of mural cells. Tissue-specific analyses of vascular structures revealed that NG2-expressing pericytes of the cardiac vasculature were disorganized in Pdgfd(-/-) mice. Furthermore, Pdgfd(-/-) mice also had a slightly elevated blood pressure. In summary, the vascular expression pattern together with morphological changes in NG2-expressing cells, and the increase in blood pressure, support a function for PDGF-D in regulating systemic arterial blood pressure, and suggests a role in maintaining vascular homeostasis

Extracellular retention of PDGF-B directs vascular remodeling in mouse hypoxia-induced pulmonary hypertension

Pulmonary hypertension (PH) is a lethal condition, and current vasodilator therapy has limited effect. Antiproliferative strategies targeting platelet-derived growth factor (PDGF) receptors, such as imatinib, have generated promising results in animal studies. Imatinib is, however, a nonspecific tyrosine kinase inhibitor and has in clinical studies caused unacceptable adverse events. Further studies are needed on the role of PDGF signaling in PH. Here, mice expressing a variant of PDGF-B with no retention motif (Pdgfbret/ret), resulting in defective binding to extracellular matrix, were studied. Following 4 wk of hypoxia, right ventricular systolic pressure, right ventricular hypertrophy, and vascular remodeling were examined. Pdgfbret/ret mice did not develop PH, as assessed by hemodynamic parameters. Hypoxia did, however, induce vascular remodeling in Pdgfbret/ret mice; but unlike the situation in controls where the remodeling led to an increased concentric muscularization of arteries, the vascular remodeling in Pdgfbret/ret mice was characterized by a diffuse muscularization, in which cells expressing smooth muscle cell markers were found in the interalveolar septa detached from the normally muscularized intra-acinar vessels. Additionally, fewer NG2-positive perivascular cells were found in Pdgfbret/ret lungs, and mRNA analyses showed significantly increased levels of Il6 following hypoxia, a known promigratory factor for pericytes. No differences in proliferation were detected at 4 wk. This study emphasizes the importance of extracellular matrix-growth factor interactions and adds to previous knowledge of PDGF-B in PH pathobiology. In summary, Pdgfbret/ret mice have unaltered hemodynamic parameters following chronic hypoxia, possibly secondary to a disorganized vascular muscularization

MicroRNA-Dependent Control of Serotonin-Induced Pulmonary Arterial Contraction

Background: Serotonin (5-HT) is considered to play a role in pulmonary arterial hypertension by regulating vascular remodeling and smooth muscle contractility. Here, arteries from mice with inducible and smooth muscle-specific deletion of Dicer were used to address mechanisms by which microRNAs control 5-HT-induced contraction. Methods: Mice were used 5 weeks after Dicer deletion, and pulmonary artery contractility was analyzed by wire myography. Results: No change was seen in right ventricular systolic pressure following dicer deletion, but systemic blood pressure was reduced. Enhanced 5-HT-induced contraction in Dicer KO pulmonary arteries was associated with increased 5-HT2A receptor mRNA expression whereas 5-HT1B and 5-HT2B receptor mRNAs were unchanged. Contraction by the 5-HT2A agonist TCB-2 was increased in Dicer KO as was the response to the 5-HT2B agonist BW723C86. Effects of Src and protein kinase C inhibition were similar in control and KO arteries, but the effect of inhibition of Rho kinase was reduced. We identified miR-30c as a potential candidate for 5-HT2A receptor regulation as it repressed 5-HT2A mRNA and protein. Conclusion: Our findings show that 5-HT receptor signaling in the arterial wall is subject to regulation by microRNAs and that this entails altered 5-HT2A receptor expression and signaling

<i>Pdgfd</i> is highly expressed in the cardiac vasculature, particularly in arterial bifurcations.

<p>Analysis of heart, whole mounts and sections from wildtype and <i>Pdgfd</i>^+/- mice. (A) Postnatal (day P4) whole mount heart showing X-gal staining (blue) (B) Adult whole mount hearts from 16 weeks old mice <i>Pdgfd</i>^+/+ and <i>Pdgfd</i>^+/- mice showing X-gal staining. (C) Magnification from (B). (D-G) Representative images of sections from heart showing X-gal staining. (D) <i>Pdgfd</i>^+/+, control. (E) Overview of <i>Pdgfd</i>^+/- heart. (F) Higher magnification of <i>Pdgfd</i>^+/- heart, strong staining in blood vessel bifurcation (arrow). (G) <i>Pdgfd</i>^+/- counterstained with hematoxylin and eosin. (H-K) Representative images of <i>Pdgfd</i>^+/- heart sections from 21 weeks old mice, showing podocalyxin (H, J) staining as a marker of endothelial cells (green) and X-gal staining (blue). (H) Artery showing X-gal co-staining with podocalyxin (upper arrow) and X-gal staining outside of podocalyxin staining (lower arrow) and (I) in the same section, artery showing X-gal without podocalyxin. (J) A vein showing limited X-gal staining (arrow), and (K) the same section, without podocalyxin staining. (L-O) Representative confocal images of a <i>Pdgfd</i>^<i>+/-</i> heart section from 16 weeks old mice showing immunofluorescent alpha-smooth muscle actin (αSMA) and enzymatic X-gal staining visualized by transmitted light. (L) Arteries showing αSMA (red) and X-gal staining (black). (M-O) Magnifications from (L), arrows pointing at black X-gal staining. (M) Arteries showing αSMA (red) and X-gal staining (black). (N) αSMA (white), no transmitted light. (O) X-gal staining (black). Scale bars 100 μm.</p

Intrauterine vertical SARS-CoV-2 infection : a case confirming transplacental transmission followed by divergence of the viral genome

A 27-year-old woman (gravida 2, para 1) was transported to the regional university hospital in gestational week (GW) 34 + 4 due to a three-day history of fever, abdominal pain and reduced foetal movements. She had developed a dry cough one day prior to the admission (Figure S1). The woman, was slightly overweight (BMI 27 kg/m2 ) but otherwise healthy. She had normal antenatal check-ups and an obstetric ultrasound at GW 32 + 2 showed a normal foetal weight deviation of +8%1

<i>Pdgfd</i> promoter-driven <i>LacZ</i> expression displays a consistent vascular localization pattern.

<p>(A) qPCR analysis on RNA from different tissues from 13 weeks old adult wild-type C57BL/6 mice. Total expression of <i>Pdgfd</i> mRNA measured as % of <i>L19</i> and normalized to adrenal gland (peak organ). (B-U) X-gal staining (blue) of frozen sections from different tissues from 21 weeks old <i>Pdgfd</i>^+/- mice, counterstained with hematoxylin. (B) Adrenal gland. (C) Adrenal cortex. (D) Adrenal medulla. (E) Aorta. (F) Aorta, higher magnification. (G) Heart. (H) Heart, myocardium, higher magnification. (I) Uterus. (J) Uterus, endometrium. (K) Testis. (L) Testis, seminiferous tubules. (M) Kidney. (N) Kidney, glomerulus and afferent arteriole. (O) Renal papilla. (P) Liver. (Q) Liver, bile duct. (R) Liver, blood vessel. (S) Pancreas, endocrine islet indicated by the dashed line. (T) Pancreatic duct (U) Pancreas, blood vessel. Scale bars 20, 50 or 100 μm. Error bars in (A) indicate standard deviation.</p