Changes in the signal transduction efficiency of senescent cells led us to compare the signaling events induced by two mitogenic agonists, platelet-derived growth factor (PDGF) and lysophosphatidic acid (LPA) in presenescent and senescent or near-senescent human diploid fibroblasts. When the changes in intracellular [Ca(2+)](i) were analyzed, both PDGF and LPA generated a rhythmic increase in [Ca(2+)](i) in presenescent cells. The frequency of calcium response was reduced and desensitized in PDGF-stimulated senescent cells, while response to a LPA-induced calcium signal was also reduced in frequency, though its magnitude was unaltered. PDGF treatment increased the fibrous actin (F-actin) level in presenescent cells but not in senescent cells in contrast to a reduced but visible increase in F-actin in LPA-treated senescent cells. The effect of PDGF on phospholipase D (PLD) activation was also reduced significantly, as a ca. 60-80% reduction of PLD activity was observed in PDGF-stimulated cells but only a little reduction in LPA-induced cells. Agonist-specific differential changes of cellular signaling events caused a differential effect on DNA synthesis after growth factor stimulation. We observed a dramatic (80-90%) reduction of [3H]thymidine incorporation into DNA in the PDGF-stimulated near-senescent cells. LPA resulted in a 2-3-fold increase in thymidine incorporation even in the near-senescent cells. These differences in the responses of senescent or near-senescent cells to PDGF- and LPA-stimulation raised questions about the differential changes of the respective signaling apparatuses induced by aging. Since PDGF signaling event was affected greatly by aging, we further examined the protein contents involved in PDGF signal transduction pathway. PDGF receptor (PDGFR), protein kinase C-alpha (PKC-alpha), phospholipase C-gamma1 (PLC-gamma1), and PLDDPDGFR were reduced with age. The reduced content of PDGFR protein may be one of the important contributors to the failure of PDGF-stimulated signal transduction in human senescent fibroblasts. Our results strongly suggest that age-dependent agonist-specific changes in signaling events might be in charge of the functional deterioration of senescent cells through imbalance of signal responses.

Platelet-derived growth factors are a family of mitogens and chemoattractants comprising of four ligand genes (A-, B-, C-, D-chains) implicated in many physiologic and pathophysiologic processes, including atherosclerosis, fibrosis and tumorigenesis. Our understanding of the molecular mechanisms, which regulate PDGF-C transcription remains incomplete. Transient transfection analysis, conventional and quantitative real-time PCR revealed the induction of PDGF-C transcription and mRNA expression in smooth muscle cells (SMCs) exposed to the peptide hormone angiotensin (ATII), which induces Egr-1. Occupancy of a G + C-rich element in the proximal region of the PDGF-C promoter was unaffected by ATII. Instead we discovered, using both nuclear extracts and recombinant proteins with EMSA and ChIP analyses, the existence of a second Egr-1-binding element located 500 bp upstream. ATII induction of PDGF-C transcription is mediated by the angiotensin type 1 receptor (AT1R) and Egr-1 activation through this upstream element. DNAzyme EDPDGF-C expression. Moreover, increased PDGF-C expression after exposure to ATII depends upon the differentiation state of the SMCs. This study demonstrates the existence of this novel ATII-AT1R-Egr-1-PDGF-C axis in SMCs of neonatal origin, but not in adult SMCs, where ATII induces Egr-1 but not PDGF-C.

It is known that platelet alpha-granule constituents including platelet-derived growth factor (PDGF), platelet factor 4 (PF4) and transforming growth factor-beta (TGF-beta) can affect megakaryocytopoiesis. Serotonin, a platelet dense granule constituent has been shown to have a mitogenic effect on fibroblasts and smooth muscle cells but whether it has the same effect on megakaryocytes remains unclear. In this study, we investigated the effect of serotonin on megakaryocytopoiesis and the possible mechanism of its effect using the mouse plasma clot culture method. The results show that: (a) serotonin significantly stimulates megakaryocyte colony formation with maximum stimulation at 100 nM; (b) enhanced action is found between serotonin and interleukin-3 (IL-3), interleukin-6 (IL-6), granulocyte macrophage-colony stimulating factor (GM-CSF), erythropoietin (EPO) and PDGF; (c) ketanserin, a 5-HT2 receptor antagonist, blocks the mitogenic effect of serotonin on megakaryocytopoiesis; and (d) Meg-01 cells (a megakaryocyte cell line) express 5-HT2 receptors. This study demonstrates that serotonin has a mitogenic effect on megakaryocytopoiesis and this effect may be mediated via the 5-HT2 receptor which is known to be coupled to G protein. It is suggested that serotonin may also be involved in the feedback control of megakaryocytopoiesis.

BACKGROUND

The present study was designed to evaluate the impact of the tyrosine kinase ligands VEGF-A/C/D, PDGF-A/B on tumor dissemination and survival in gastric cancer. This is the first study analyzing all these parameters in a homogeneous patient population undergoing surgery.

METHODS

The expression pattern of VEGF-A/C/D and PDGF-A/B was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) in 69 samples of human gastric adenocarcinoma and correlated with tumor stage and survival.

RESULTS

Expression of the ligand VEGF-D significantly correlated with distant metastatic disease (P=0.00001) but not with patient survival. However, VEGF-A inversely correlated with M1 and grading, PDGF-A inversely correlated with pT and pN category. In contrast, VEGF-C and PDGF-B did not have an impact on clinicopathological parameters.

CONCLUSIONS

The ligand VEGF-D, rather than the other ligands or tyrosine kinase receptors analyzed, is associated with progressive disease in gastric cancer patients undergoing surgery. The VEGF-D ligand might be a helpful marker indicating disseminated disease, and targeting VEGF-D may be a potential therapeutic strategy, although limitations imposed by the selected sample population have to be considered critically.

OBJECTIVE

Although clopidogrel does not cause gastric mucosal injury, it does not prevent peptic ulcer recurrence in high-risk patients. We explored whether clopidogrel delays gastric ulcer healing via inhibiting angiogenesis and to elucidate the possible mechanisms.

METHODS

Gastric ulcers were induced in Sprague Dawley rats, and ulcer healing and angiogenesis of ulcer margin were compared between clopidogrel-treated rats and controls. The expressions of the proangiogenic growth factors and their receptors including basic fibroblast growth factor (bFGF), bFGF receptor (FGFR), vascular endothelial growth factor (VEGF), VEGFR1, VEGFR2, platelet-derived growth factor (PDGF)A, PDGFB, PDGFR A, PDGFR B, and phosphorylated form of mitogenic activated protein kinase pathways over the ulcer margin were compared via western blot and reverse transcription polymerase chain reaction. In vitro, human umbilical vein endothelial cells (HUVECs) were used to elucidate how clopidogrel inhibited growth factors-stimulated HUVEC proliferation.

RESULTS

The ulcer sizes were significantly larger and the angiogenesis of ulcer margin was significantly diminished in the clopidogrel (2 and 10 mg/kg/d) treated groups. Ulcer induction markedly increased the expression of phosphorylated form of extracellular signal-regulated kinase (pERK), FGFR2, VEGF, VEGFR2, and PDGFRA when compared with those of normal mucosa. Clopidogrel treatment significantly decreased pERK, FGFR2, VEGF, VEGFR2, and PDGFRA expression at the ulcer margin when compared with those of the respective control group. In vitro, clopidogrel (10(-6)M) inhibited VEGF-stimulated (20 ng/mL) HUVEC proliferation, at least, via downregulation of VEGFR2 and pERK.

CONCLUSIONS

Clopidogrel inhibits the angiogenesis of gastric ulcer healing at least partially by the inhibition of the VEGF-VEGFR2-ERK signal transduction pathway.

As previous studies showed, PDGF-AA exerts a poor mitogenic effect on vascular smooth muscle cells. Simultaneous addition of insulin-like growth factor 1 (IGF-1), itself also poorly mitogenic, led to a significant increase in [3H]thymidine incorporation into the cell DNA as well as a strong increase in cell number. To explain the synergistic effect of PDGF-AA and IGF-1 on VSMC proliferation, we describe the effects of the two growth factors on distinct intracellular signals: on the activation of the signal proteins mitogen-activated protein kinase (MAPK) isoforms p42 and p44 and on the protein kinase C (PKC) isoforms alpha, delta, and epsilon, and on the induction of the transcription factor c-fos. PDGF-AA strongly activated the MAPK isoforms and PKC delta as well as the induction of c-fos. In contrast, IGF-1 exerted no effect on the signals induced by PDGF-AA, but strongly activated PKC epsilon isoform. Comparing this signal pattern to the one of the mitogenically potent PDGF isoform PDGF-BB, we found that PDGF-BB activated all of the signal proteins investigated.

In Rat-1 fibroblasts, v-Src causes a profound remodelling of cortical actin cytoskeleton. This transformation includes membrane ruffling, a hallmark of the leading edge in migrating cells, and results from activation of phosphoinositide 3-kinase (PI 3-kinase), phospholipase C (PLC) and phospholipase D (PLD). We therefore reexamined whether motility is constitutively triggered by v-Src and studied whether this response is controlled by the same signalling pathway. The study was performed using Rat-1/tsLA29 and MDCK/tsLA31 cells, each harbouring a different thermosensitive v-Src kinase, active at 34 degrees C but inactivated at 40 degrees C. In both cell lines, overnight v-Src activation induced transformation and accelerated spontaneous motility by approximately twofold, as evidenced by wound-healing assay and by single-cell track, time-lapse recording in Dunn chambers. Inhibitors of PI 3-kinase, PLC and PLD selectively abrogated acceleration of motility by v-Src. Since mechanisms that co-ordinate spontaneous, as distinct from oriented, cell migration are separable, we further analysed in Dunn chambers chemotactic response of Rat-1/tsLA29 cells to PDGF and of MDCK/tsLA31 cells to EGF. In both cases, v-Src decreased the steady-state level of growth factor receptors at the cell surface twofold, and abrogated movement directionality at comparable level of occupancy as in non-transformed cells. The burst of pinocytosis in response to growth factors was also abolished by v-Src. Altogether, these results indicate that v-Src triggers motility in a PI 3-kinase-, PLC- and PLD-dependent manner, but abrogates directionality by suppressing polarised signalling downstream of growth factor receptors.

Vascular smooth muscle cell (VSMC) chemotaxis is fundamental to atherosclerosis and intimal hyperplasia. An increase in intracellular Ca2+ [Ca2+]i is an important signal in chemotaxis, but the role of L-type calcium channels (CaV1.2) in this response in human vascular smooth muscle cells (hVSMC) has not been examined. hVSMC were grown from explant cultures of saphenous vein. Confluent hVSMC at passage 3 were studied after culture in medium containing 15% foetal calf serum (FCS) (randomly cycling) or following serum deprivation for up to 7 days. Smooth muscle alpha-actin was measured by immunoblotting and immunofluorescence microscopy. [Ca2+]i was measured using fura 2 fluorimetry. Chemotaxis was measured using a modified Boyden chamber technique and cell attachment to gelatin-coated plates was also quantified. The number and affinity of dihydropyridine-binding sites was assessed using [5-methyl-3H]PN 200-110 binding. In randomly cycling cells, the calcium channel agonist, Bay K 8644a and 100 mM KCl did not affect [Ca2+]i. In addition, the rise in [Ca2+]i induced by platelet-derived growth factor-BB (PDGF) was unaffected by the CaV1.2 antagonists, amlodipine and verapamil. In randomly cycling cells amlodipine did not affect PDGF-induced migration. In serum-deprived cells, smooth muscle alpha-actin was increased and Bay K 8644a and 100 mM KCl increased [Ca2+]i. PDGF-induced rises in [Ca2+]i were also inhibited by amlodipine and verapamil. The ability of Bay K 8644a to increase [Ca2+]i and verapamil to inhibit PDGF-induced rises in [Ca2+]i was evident within 3 days after serum withdrawal. In serum-deprived hVSMC Bay K 8644a induced chemotaxis and amlodipine inhibited PDGF-induced migration. Cell attachment in the presence of PDGF was unaffected by amlodipine in either randomly cycling or serum-deprived hVSMC. Serum withdrawal was associated with a decrease in the maximum number of dihydropyridine-binding sites (B(max)) and a decrease in affinity (K(D)). Serum deprivation of hVSMC results in increased expression of smooth muscle alpha-actin, a marker of more differentiated status, and increased [Ca2+]i responses and chemotaxis mediated by CaV1.2. These observations may have important implications for understanding the therapeutic benefits of calcium channel antagonists in cardiovascular disease.

Cultivated arterial smooth muscle cells were used to study the effects of platelet-derived growth factor (PDGF) on polyamine synthesis and of polyamine synthesis inhibitors on initiation of DNA synthesis by PDGF. Quiescent (serum-starved) cells showed no detectable activity of ornithine decarboxylase (ODC), the first and overall rate-limiting enzyme in polyamine synthesis, and had low levels of the polyamines putrescine, spermidine and spermine. PDGF caused a marked increase in ODC activity, with a peak after 6 h followed by a rapid decline. The rise in enzyme activity was blocked by actinomycin D and cycloheximide, suggesting control at the transcriptional and translational levels, alpha-Difluoromethylornithine (DFMO), a catalytic and irreversible inhibitor of ODC, prevented the appearance of enzyme activity. The cellular content of putrescine increased distinctly in response to PDGF, with a peak after 8 h, abolished by simultaneous treatment with DFMO. In contrast, the concentrations of spermidine and spermine changed but little within the 20-h period examined here. DFMO inhibited the initiation of DNA synthesis without affecting the length of the prereplicative lag phase and was fully active if added within 4 h after PDGF. The effect of DFMO on DNA synthesis was counteracted by addition of polyamines to the culture medium and exogenous polyamines were also found to stimulate DNA synthesis in PDGF-free medium (spermine greater than spermidine greater than putrescine). It is concluded that induction of ODC and putrescine synthesis are integral parts in the mitogenic response of arterial smooth muscle cells to PDGF.

The present study was undertaken to clarify the relationship between c-fos and c-jun protooncogene expression and the differentiation and/or proliferation of osteoblasts, using osteoblast-like MC3T3-E1 (E1) cells. c-fos mRNA was barely detectable, whereas c-jun mRNA was constitutively expressed in E1 cells after serum deprivation for 24-72 h. When serum was added, a rapid and transient induction of c-fos and c-jun mRNAs was observed. The c-fos and c-jun mRNAs reached peak levels at 30 minutes, with a rapid disappearance of c-fos mRNA within 3 h and a much slower decrease in c-jun mRNA. The addition of serum together with cycloheximide, an inhibitor of protein synthesis, resulted in the superinduction of both c-fos and c-jun mRNAs. Among various growth factors, PDGF, EGF, and bFGF mimicked the serum effect, whereas IGF-I and TGF-beta failed to induce c-fos and c-jun mRNA. The effects of PDGF, EGF, and bFGF were completely abolished by pretreatment with actinomycin D, an inhibitor of RNA synthesis, suggesting a transcriptional mechanism. Nuclear runoff experiments showed that the transcription rate of c-fos and c-jun protooncogenes was increased by serum and growth factors. The effects of PDGF, EGF, and bFGF were inhibited by H-7 or staurosporine, inhibitors of protein kinase C (PKC), but not by HA1004 with a much weaker inhibitory activity, suggesting the involvement of PKC for the activation of the protooncogenes.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet-derived growth factors (PDGF) play a major role in pericyte recruitment in tumor capillaries. Pericytes are required for proper vessel development, and contribute to tumor angiogenesis by promoting stabilization and maturation of newly formed vessels. To investigate the effects of pericyte coverage on tumor vessel morphology and function in vivo, tumors derived from B16 melanoma cells transfected with either control plasmid (B16/ctr) or plasmid encoding full-length PDGF-BB (B16/PDGF), the latter previously shown to have enhanced blood vessel pericyte coverage and an increased tumor growth rate, were assessed using histopathological methods, Hoechst 33342-based perfusion analyses, and two noninvasive susceptibility magnetic resonance imaging (MRI) methods. Susceptibility-contrast MRI, incorporating the use of ultrasmall superparamagnetic iron oxide particles, revealed a significant (p < 0.05) reduction in vessel size index (R(v)) of B16/PDGF tumors, and which was validated histologically by the presence of significantly smaller (p < 0.001), more punctate blood vessels identified by fluorescence microscopy of the perfusion marker Hoechst 33342. Intrinsic-susceptibility MRI was used to measure the transverse MRI relaxation rate R(2)*, sensitive to changes in endogenous paramagnetic [deoxyhaemoglobin], and used to probe for vascular maturation and function. Hypercapnia (5% CO(2)/95% air) induced a negligible Delta R(2)* response in the B16/ctr and B16/PDGF tumors. In contrast, hyperoxia (5% CO(2)/95% O(2)) induced a significantly greater R(2)* reduction in the B16/PDGF tumors (p < 0.02). Together the susceptibility MRI-derived biomarkers reveal novel pericyte-dependent changes in the morphology and function of the perfused tumor vasculature in vivo.

Exposure to mineral dusts is associated with the development of chronic airflow obstruction, probably mediated in part by dust-induced fibrosis of the small airways. To investigate the mechanism of fibrosis, we exposed rat tracheal explants to amosite asbestos, iron oxide, or titanium dioxide. Explants were then maintained in air organ culture, and the expression of genes encoding for various mediators and matrix components assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). At 7 d, all dusts produced significant increases in platelet-derived growth factor-A (PDGF-A) and transforming growth factor-beta1 (TGF-beta1) gene expression compared with control; asbestos and titanium dioxide produced increases in PDGF-B, and titanium dioxide increased TGF-alpha expression. Only asbestos caused increases in procollagen expression. No dust increased expression of tumor necrosis factor-alpha (TNF-alpha), fibronectin, or tropoelastin. Elevations in these factors coincided temporally with transport of particles into the epithelium and then to the subepithelial space. By in situ hybridization, TGF-beta gene expression was found in both the epithelium and subepithelial (interstitial) space, and PDGF-B and procollagen gene expression in the subepithelial space. Chemical analysis showed a small increase in hydroxyproline, a measure of collagen content, in asbestos-treated explants. We conclude that mineral dusts can induce airway wall fibrosis by directly upregulating proliferative and fibrogenic mediators as well as matrix components in the airway epithelium and interstitium, and that neither airspace nor circulating inflammatory cells are required for these effects. Different mineral dusts produce different patterns of reaction.

BACKGROUND

Knock-in mice (gp130F759) with a Y759F point mutation in gp130, a signal transducing receptor subunit shared by members of the IL-6 cytokine family, show sustained activation of STAT3, enhanced acute-phase or immune responses, and autoimmune arthritis. We conducted a detailed analysis of collagen-induced arthritis (CIA) in gp130F759 with a DBA/1J background (D/J.gp130F759).

METHODS

We backcrossed gp130F759 to C57BL/6 and DBA/1J, and compared the pathologic changes, including occurrence of arthritis, in the two distinct genetic backgrounds. We analyzed CIA in D/J.gp130F759 and investigated the effects of methotrexate (MTX) on CIA.

RESULTS

C57BL/6 background gp130F759 mice, but not D/J.gp130F759, spontaneously developed polyarthritis and glomerulonephritis. On the other hand, keratitis of the eyes only developed in D/J.gp130F759, indicating the influence of genetic background on disease development in gp130F759 mice. Resistance of the DBA/1J background against spontaneous arthritis urged us to examine CIA in D/J.gp130F759. CIA in D/J.gp130F759 was more severe, with greater bone destruction, than the control mice. After collagen immunization, splenomegaly and serum levels of rheumatoid factor and anti-DNA antibody were augmented in D/J.gp130F759. Bio-Plex analysis of serum cytokines revealed increased IL-12p40 and PDGF-BB before immunization, and increased levels of IFN-gamma, IL-17, TNF-alpha, IL-9, and MIP-1beta 8 days after the booster dose. IL-6 and PDGF-BB in D/J.gp130F759 showed distinct kinetics from the other cytokines; higher levels were observed after arthritis development. MTX partially attenuated the development of arthritis and inhibited bone destruction in D/J.gp130F759, with reduction of anti-type II collagen antibody levels, suggesting that MTX mainly affects antigen-specific immune responses in CIA.

CONCLUSIONS

The Tyr-759 point mutation of the IL-6 family cytokine receptor subunit, gp130, caused autoimmune disease, and this was also influenced by the genetic background. CIA in D/J.gp130F759 is useful for evaluating drugs in a relatively short period because sustained activation of STAT3 may enhance the disease symptoms.

BACKGROUND

Platelet-derived growth factor (PDGF) is a central mediator of mesangioproliferative glomerulonephritis (GN). In experimental mesangioproliferative GN, PDGF-DD serum levels, unlike PDGF-BB, increased up to 1000-fold.

METHODS

We assessed disease activity in 72 patients with GN, established a novel PDGF-D ELISA and then determined their PDGF-DD levels. In parallel, we studied renal PDGF-DD mRNA expression by RT-PCR.

RESULTS

PDGF-DD serum levels in patients with IgA nephropathy (IgAN) were significantly higher (1.67 +/- 0.45 ng/ml) and in patients with lupus nephritis significantly lower (0.66 +/- 0.86 ng/ml) compared to healthy controls (1.17 +/- 0.46 ng/ml), while patients with focal segmental glomerulosclerosis, membranous GN and ANCA-positive vasculitis did not differ from controls. The subgroup of IgAN patients with elevated PDGF-DD levels (27% of samples) did not differ in their clinical features from those with normal PDGF-DD levels. In IgAN patients with repetitive PDGF-DD determinations, most exhibited only minor fluctuations of serum levels over time. Intrarenal PDGF-DD mRNA expression did not differ between controls and patients, suggesting an extrarenal source of the elevated PDGF-DD in IgAN.

CONCLUSIONS

Serum PDGF-DD levels were specifically elevated in patients with IgAN, in particular in those with early disease, i.e. preserved renal function. Our data support the rationale for anti-PDGF-DD therapy in mesangioproliferative GN.

Growth failure in zinc-deficient animals is associated with decreased DNA synthesis; zinc deprivation of 3T3 cells, by use of diethylenetrinitrilopentaacetate (DTPA), impairs thymidine incorporation when the cells are stimulated with fetal bovine serum (FBS). The purpose of this study was to determine the step of cell cycle progression that is affected by zinc deprivation. Swiss murine 3T3 cells were cultured for 3 d in complete media and then for 2 d in low serum media. Cells were then placed in serum-free media and stimulated in sequence with platelet-derived growth factor (PDGF; 3 h), epidermal growth factor (EGF; 0.5 h) and insulin-like growth factor-I (IGF-I; 16 h). The combination of growth factors stimulated thymidine incorporation to the same extent as 10% FBS, and DTPA or EDTA (0.6 mmol/L) inhibited thymidine incorporation. Inhibition was prevented by addition of zinc, but not calcium, iron or cadmium (0.4 mmol/L). When DTPA was present during all stages with no addition of zinc, or zinc added during the competency-priming (PDGF and EGF) step, the IGF-I step, or both steps, the zinc effect occurred at the IGF-I step. Zinc addition 4 h before the measurement of thymidine incorporation had no ameliorative effect, but the presence of zinc during the prior 12 h increased incorporation. Thus zinc exerts its major effect on DNA synthesis during the IGF-I stimulatory phase of the cell cycle. The total zinc concentration of 3T3 cells treated with DTPA for 16 h was not different from that of untreated cells; hence only a small compartment of the cell is affected by DTPA.

BACKGROUND

Targeted therapies in renal cell carcinoma can have different effects on primary and metastatic tumors. To pave the way for predictive biomarker development, we assessed differences in expression of targets of currently approved drugs in matched primary and metastatic specimens from 34 patients.

METHODS

Four cores from each site were embedded in tissue microarray blocks. Expression of B-Raf, C-Raf, cKIT, FGF-R1, HIF-2α, mTOR, PDGF-Rβ, VEGF-R1, VEGF-R2, VEGF-R3, VEGF, VEGF-B, VEGF-C, VEGF-D, MEK1, and ERK1/2 was studied using a quantitative immunofluorescence method.

RESULTS

No significant differences were observed in global expression levels in primary and metastatic renal cell carcinoma tumors, with the exception of MEK, which had higher expression in metastatic than primary specimens. Similarly, more ki67 positive cells were seen in metastatic specimens. Correlations between marker expression in primary and metastatic specimens were variable, with the lowest correlation seen for FGF-R1 and VEGF-D. There were no significant differences in the degree of heterogeneity in primary versus metastatic tumors.

CONCLUSIONS

Expression of most of the studied markers was similar in primary and metastatic renal cell carcinoma tumors, suggesting that predictive biomarker testing for these markers can be conducted on either the primary or metastatic tumors for most markers.

Coronavirus disease 2019 (COVI<em>D</em>-19) is associated with considerable morbidity and mortality. The number of confirmed cases of infection with SARS-CoV-2, the virus causing COVI<em>D</em>-19 continues to escalate with over 70 million confirmed cases and over 1.6 million confirmed deaths. Severe-to-critical COVI<em>D</em>-19 is associated with a dysregulated host immune response to the virus, which is thought to lead to pathogenic immune dysregulation and end-organ damage. Presently few effective treatment options are available to treat COVI<em>D</em>-19. Leronlimab is a humanized IgG4, kappa monoclonal antibody that blocks C-C chemokine receptor type 5 (CCR5). It has been shown that in patients with severe COVI<em>D</em>-19 treatment with leronlimab reduces elevated plasma IL-6 and chemokine ligand 5 (CCL5), and normalized C<em>D</em>4/C<em>D</em>8 ratios. We administered leronlimab to 4 critically ill COVI<em>D</em>-19 patients in intensive care. All 4 of these patients improved clinically as measured by vasopressor support, and discontinuation of hemodialysis and mechanical ventilation. Following administration of leronlimab there was a statistically significant decrease in IL-6 observed in patient A (p=0.034) from day 0-7 and patient <em>D</em> (p=0.027) from day 0-14. This corresponds to restoration of the immune function as measured by C<em>D</em>4+/C<em>D</em>8+ T cell ratio. Although two of the patients went on to survive the other two subsequently died of surgical complications after an initial recovery from SARS-CoV-2 infection.

Keywords: ACE2, angiotensin-converting enzyme 2; ALT, alanine aminotransferase; ARDS, acute respiratory distress syndrome; AST, aspartate aminotransferase; Acute respiratory distress syndrome (ARDS); BID, bis in die (twice a day); CCL2, chemokine C–C motif ligand 2; CCL3, chemokine C–C motif ligand 3; CCL4, chemokine C–C motif ligand 4; CCL5, chemokine C–C motif ligand 5; CCR1, C–C chemokine receptor type 1; CCR5, C–C chemokine receptor type 5; CDC, Centers for Disease Control; CK, creatine kinase; COPD, chronic obstructive pulmonary disease; COVID-19, coronavirus disease 2019; CRP, C-reactive protein; CXCL10, chemokine C-X-C motif ligand 10; CXCL2, chemokine C-X-C motif ligand 2; Coronavirus disease 2019 (COVID-19); DPP4, dipeptidyl peptidase-4; DVT, deep vein thrombosis; EDTA, ethylenediaminetetraacetic acid; FDA, Food and Drug Administration; Fi02, fraction of inspired oxygen, IgG4; Hydroxychloroquine, HLH; Leronlimab (PRO 140); Middle East respiratory syndrome coronavirus, MIG; National Early Warning Score, NK; RO, receptor occupancy; RT–PCR, reverse transcriptase polymerase chain reaction; SARS-CoV, severe acute respiratory syndrome coronavirus; SARS-CoV-2; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; T-reg RO, regulatory T cells – receptor occupancy; TGF- α, transforming growth factor alpha; TNF-α, tumor necrosis factor alpha; TNF-β, tumor necrosis factor beta; Tregs, regulatory T cells; VEGF-A, vascular endothelial growth factor A; WBC, white blood cell; WHO, World Health Organization; eIND, emergency investigational new drug application; hemophagocytic lymphohistiocytosis, HTN; hypertension, ICU; immunoglobulin G4, HCQ; intensive care unit, IL-1β; interferon gamma, IL-6; interferon gamma-inducible protein (IP) 10 or CXCL10, LOA; interleukin 1 beta, IFN-ƴ; interleukin 6, IP-10; letter of authorization, MCP; macrophage Inflammatory Proteins 1-alpha, MIP-1β; macrophage Inflammatory Proteins 1-beta, N/A; macrophage colony stimulating factor, MDC (CCL22); macrophage colony-stimulating factor encoded by the CCL22 gene, MERS-CoV; monocyte chemoattractant protein, M-CSF; monokine induced by IFN-γ (interferon gamma), MIP-1α; natural killer, OSA; not applicable, NEWS2; obstructive sleep apnea, PDGF-AA; per os (taken by mouth), RANTES; platelet-derived growth factor AA, PDGF-AA/BB; platelet-derived growth factor AA/BB, PEEP; positive end-expiratory pressure, PNA; pulmonary nodular amyloidosis, po; regulated on activation, normal T expressed and secreted (also known as CCL5).

Platelet-derived growth factor (PDGF) is a potent moderator of soft tissue repair through induction of the inflammatory phase of repair and subsequent enhanced collagen deposition. We examined the effect of recombinant BB homodimer PDGF (rPDGF-BB) applied to rat craniotomy defects, treated with and without bovine osteogenin (OG), to see if bone regeneration would be stimulated. Implants containing 0, 20, 60, or 200 micrograms rPDGF-BB, reconstituted with insoluble rat collagenous bone matrix containing 0, 30, or 150 micrograms OG, were placed into 8-mm craniotomies. After 11 d, 21 of the 144 rats presented subcutaneous masses superior to the defect sites. The masses, comprised of serosanguinous fluid encapsulated by fibrous connective tissue, were larger and occurred more frequently in rats treated with 200 micrograms rPDGF-BB, and were absent in rats not treated with rPDGF-BB. The masses underwent resorption within 28 d after surgery. OG (2-256 micrograms) caused a dose-dependent increase in radiopacity and a marked regeneration of calcified tissue in a dose-dependent fashion within defect sites. However, OG-induced bone regeneration was inhibited 17-53% in the presence of rPDGF-BB. These results suggest that rPDGF-BB inhibited OG-induced bone regeneration and stimulated a soft tissue repair wound phenotype and response.

CCAAT/enhancer-binding protein (C/EBP)-binding motifs have been identified in the promoter regions of interleukin (IL)-6, tumor necrosis factor-alpha, and platelet-derived growth factor-alpha receptor (PDGFalphaR). Recently, peroxisome proliferator-activated receptors (PPARs) have been suggested to be important immunomodulatory mediators. Although many studies have demonstrated that the interaction between C/EBPs and PPARs plays a central role in lipid metabolism, expression and function of these factors are unknown in vascular smooth muscle cells (VSMCs). In the present study, we clarified a functional relationship between C/EBPs and PPARgamma in the regulation of IL-1beta-induced PDGFalphaR expression in VSMCs. PPARgamma activators, troglitazone and 15-deoxy-Delta(12,14)-prostaglandin J(2), inhibited IL-1beta-induced PDGFalphaR expression and suppressed PDGF-induced proliferation activity of VSMCs. Electromobility shift and supershift assays for a C/EBP motif in the PDGFalphaR promoter region revealed that PPARgamma activators suppressed IL-1beta-induced DNA binding activity of C/EBPdelta and beta. PPARgamma activators also suppressed IL-1beta-induced C/EBPdelta expression. In contrast, overexpression of C/EBPdelta reversed the suppressive effect of PPARgamma activators on PDGFalphaR expression almost completely. From these results, we conclude that the inhibitory effect of PPARgamma activators on PDGFalphaR expression is mainly mediated by C/EBPdelta suppression. Regulation of C/EBPdelta by PPARgamma activators probably plays critical roles in modulating inflammatory responses in the arterial wall.

Recent molecular cloning studies have suggested the presence of at least two beta4Gal transferase genes (beta4GalT-V and beta4GalT-VI) that may encode lactosylceramide synthase but whether they are functional in vivo and whether they mediate growth factor induced phenotypic change such as cell proliferation is not known. Our previous studies lead to the suggestion that various risk factors in atherosclerosis such as oxidized LDL, shear stress, nicotine, tumor necrosis factor-alpha converge upon LacCer synthase to induce critical phenotypic changes such as cell proliferation and cell adhesion. However, whether platelet-derived growth factor also recruits LacCer synthase in mediating cell proliferation is not known. Here we have employed a Chinese hamster ovary mutant cell line Pro(-)5Lec20 to determine whether this enzyme physiologically functions to mediate cell proliferation. We show that PDGF stimulates the activity of UDP galactose:glucosylceramide, beta1,4galactosyltransferase. The activity of LacCer synthase increased about 2.5 fold within 2.5-5 min of incubation with PDGF in both wild type and Pro(-)5Lec20 cells. Concomitantly, there was an increase in the generation of superoxide radicals, p44MAPK phosphorylation and cell proliferation in CHO cells. D-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a potent inhibitor of GlcCer synthase/LacCer synthase impaired PDGF mediated induction of LacCer synthase activity, superoxide generation, p44 MAPK activation and cell proliferation in Pro(-)5Lec20 cells. PDGF-induced superoxide generation was also mitigated by the use of diphenylene iodonium; an inhibitor of NADPH oxidase activity that is required for superoxide generation. This inhibition was bypassed by the addition of lactosylceramide. Thus, beta4GalT-V gene produces a bona fide LacCer synthase that can function in vivo to generate LacCer. Moreover, this enzyme alone can mediate PDGF induced activation of a signal transduction cascade involving superoxide generation, p44MAPK activation, phosphorylation of Akt and cell proliferation.

Pulmonary arterial hypertension (PAH) is characterized by excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs). MicroRNAs have been implicated in the regulation of cell proliferation and might be implicated in the etiology of PAH. Data from in vivo and in vitro cell culture models showed that hypoxia inhibits microRNA-30c (miR-30c) expression in PASMCs. Inhibition of miR-30c by either hypoxia or AMO-30c results in PASMC proliferation (cell viability, 5-bromo-2-deoxyuridine (BrdU) incorporation, proliferating cell nuclear antigen, Ki67, and tubulin polymerization) and the inhibition of apoptosis (cell cycle progression, Cyclin A and Cyclin D, and TUNEL staining). Moreover, down-regulation of miR-30c also results in the phenotype switch from contractile to synthetic PASMC (SM22α and Calponin, osteopontin expression, and wound healing assay). In contrast, these effects were reversed by the application of an miR-30c mimetic under hypoxic conditions. Mechanically, miR-30c inhibited the platelet-derived growth factor receptor β (PDGFRβ) expression by directly binding to the 3' untranslated region of PDGFRβ mRNA (luciferase reporter assays, and PDGFRβ-masking antisense oligodeoxynucleotides). Pharmacological inhibition of PDGFR by AG-1296 displayed similar effects to the miR-30c mimetic. These data suggest that the down-regulation of miR-30c accounts for the up-regulation of PDGFRβ expression, and subsequent activation of PDGF signaling results in the hypoxia-induced PASMC proliferation and phenotype switching. Therefore, increasing miR-30c expression levels could be explored as a potential new therapy for hypoxia-induced PAH.

BACKGROUND

The Duffy antigen receptor for chemokines (DARC) shows high affinity binding to multiple inflammatory CC and CXC chemokines and is expressed by erythrocytes and endothelial cells. Recent evidence suggests that endothelial DARC facilitates chemokine transcytosis to promote neutrophil recruitment. However, the mechanism of chemokine endocytosis by DARC remains unclear.

RESULTS

We investigated the role of several endocytic pathways in DARC-mediated ligand internalization. Here we report that, although DARC co-localizes with caveolin-1 in endothelial cells, caveolin-1 is dispensable for DARC-mediated (125)I-CXCL1 endocytosis as knockdown of caveolin-1 failed to inhibit ligand internalization. (125)I-CXCL1 endocytosis by DARC was also independent of clathrin and flotillin-1 but required cholesterol and was, in part, inhibited by silencing Dynamin II expression.(125)I-CXCL1 endocytosis was inhibited by amiloride, cytochalasin D, and the PKC inhibitor Gö6976 whereas Platelet Derived Growth Factor (PDGF) enhanced ligand internalization through DARC. The majority of DARC-ligand interactions occurred on the endothelial surface, with DARC identified along plasma membrane extensions with the appearance of ruffles, supporting the concept that DARC provides a high affinity scaffolding function for surface retention of chemokines on endothelial cells.

CONCLUSIONS

These results show DARC-mediated chemokine endocytosis occurs through a macropinocytosis-like process in endothelial cells and caveolin-1 is dispensable for CXCL1 internalization.

It has recently been demonstrated that some members of the 14-3-3 protein family play an important role in signal transduction leading to cellular proliferation. We have previously shown that expression of 14-3-3gamma is induced by growth factors in human vascular smooth muscle cells (VSMC). In this study, we cloned the human homolog of 14-3-3gamma and observed many potential phosphorylation sites, suggesting the potential for post-translational modification. In VSMC treated with platelet-derived growth factor (PDGF), 14-3-3gamma protein was expressed and phosphorylated in an activation-dependent manner. Platelet-derived growth factor-induced phosphorylation could be inhibited by phosphokinase C (PKC) inhibitory compounds, and 14-3-3gamma could be phosphorylated in the absence of PDGF by compounds that activate PKC. We also demonstrated interaction between 14-3-3gamma and several PKC isoforms (alpha, beta, gamma, theta, and delta), implicating these PKC family isoforms as the kinases responsible for PDGF-induced 14-3-3gamma phosphorylation. We found that 14-3-3gamma interacted with the signal transduction protein Raf-1, suggesting that 14-3-3gamma provides a link between this protein and PKC. Thus, 14-3-3gamma may represent a signal transduction protein that is regulated transcriptionally and post-transcriptionally by growth factors.

Macroautophagy/autophagy is one of the major responses to stress in eukaryotic cells and is implicated in several pathological conditions such as infections, neurodegenerative diseases and cancer. Interestingly, cancer cells take full advantage of autophagy both to support tumor growth in adverse microenvironments and to oppose damages induced by anti-neoplastic therapies. Importantly, different human oncogenes are able to modulate this survival mechanism to support the transformation process, ultimately leading to 'autophagy addiction'. Still, oncogenic signaling events, impinging on the control of autophagy, are poorly characterized, limiting our possibilities to take advantage of these mechanisms for therapeutic purposes. Here, we screened a library of activated kinases for their ability to stimulate autophagy. By this approach, we identified novel potential regulators of the autophagic process and, among them, the IKBKE oncogene. Specifically, we demonstrate that this oncoprotein is able to stimulate autophagy when overexpressed, an event frequently found in breast tumors, and that its activity is strictly required for breast cancer cells to support the autophagic process. Interestingly, different oncogenic pathways typically involved in breast cancer, namely ERBB2 and PI3K-AKT-MTOR, also rely on IKBKE to control this process. Ultimately, we show that IKBKE-dependent autophagy is necessary for breast cancer cell proliferation, suggesting an important supporting role for this oncogene and autophagy in these tumors.

BACKGROUND

AAK1: AP2 associated kinase 1; AMPK: 5'-prime-AMP-activated protein kinase; AKT1: AKT serine/threonine kinase 1; BAF: bafilomycin A1; CA: constitutively activated; CDK17: cyclin dependent kinase 17; CDK18: cyclin dependent kinase 18; CHUK: conserved helix-loop-helix ubiquitous kinase; EGF: epidermal growth factor; ERBB2: erb-b2 receptor tyrosine kinase 2; FGF: fibroblast growth factor; FM: full medium; GALK2: galactokinase 2; IKBKB: inhibitor of nuclear factor kappa B kinase subunit beta; IKBKE: inhibitor of nuclear factor kappa B kinase subunit epsilon; IKK: IκB kinase complex; KD: kinase dead; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPK1: mitogen-activated protein kinase 1; MAPK15: mitogen-activated protein kinase 15; MTORC1: mammalian target of rapamycin kinase complex 1; myr: myristoylation/myristoylated; NFKBIA: NFKB inhibitor alpha; PDGF: platelet derived growth factor; PFKL: phosphofructokinase, liver type; PRKAA1: protein kinase AMP-activated catalytic subunit alpha 1; PRKCD: protein kinase C delta; SQSTM1: sequestosome 1; TBK1: TANK binding kinase 1; TNBC: triple-negative breast cancer; TSC2: TSC complex subunit 2; WB: western blot; WT: wild-type.