Targeting the unstable atherosclerotic plaque : diagnostic and therapeutic implications Segers, F.M.E.
17
0
0
Hele tekst
(2) Chapter. 4.
(3)
(4) . Filip M.E. Segers1 ! " 1 # $$ %1, Claudia Peralta1, %$ $ & "1 ' " 1 $($ " 1,2. 1 &
(5) " ( )
(6) ( * &+ ( - 2
(7) + & ! ! * &+ ! - . /! 0.
(8) Part I: Targeting the Unstable Atherosclerotic Plaque. ABSTRACT. Background 3
(9) 4
(10) + 5 6$ $ . atherogenesis indicates that chemokines represent promising new targets
(11) & $ + 7 / 0 . & 8 &
(12)
(13) 4 + 6$ Methods and Results 3 7 9. +
(14) (( ) 5+ + & . $ ( 7 +'
(15)
(16)
(17) & 5+ 7 + $ !
(18) / ;< !!=>0 7 + &+
(19) 7 &#! 7 7 &+$ ?
(20) +
(21) @ B > (D (@ B (=G 7 8 + 7
(22) = > (== 5 6 $ 7 8 + 6
(23) 5 & 8
(24) 5 ) 6
(25) (D (@ 5
(26) @ 5
(27) B (=G (==$ Conclusions 3
(28) (( ) mice is characterized 5+ & 4
(29) 5+ 8
(30) @ B > 5 = >$ H . 7 5 6 & + @ .
(31) 6 $. 92.
(32) Targeting Chemokine Receptors during Atherogenesis. INTRODUCTION. 93. Chapter 4. & + 5 4.
(33)
(34) + ensuing cardiovascular disorders= D$ 6
(35) 4 + + 5 +
(36) & 5 and ruptured plaques2$ J +
(37) 4 5 + 6 . $ - + 5
(38) > and adhesion molecules5 & 5
(39) 5
(40) chemo + kines / 0 7 + ) capacities, i.e. the chemokines; L. Chemokines interact via dedicated chemokine N /N 0 L 5 7 5+
(41) + +$ ?& GO & + 5 7 Q + '
(42) 8 + 7 &+ + 5 $ +
(43)
(44) 8. ! + & & .
(45) & $
(46) GO & & 4 +
(47) 5 59 7 10. Chemokines were seen to be + 6 $ N 8
(48) 6
(49) atherosclerosis prone ApoE ) & .
(50) ! = @ ! =Y ! =Z
(51) 6 7
(52) ' 7 6 == =D. For ! = ! @ 6 7 5+ in serum levels=D. Genetic as well as pharmacological intervention studies have established a
(53) + & & $ #
(54) 5 in this regard are CCR2=>
(55) /
(56) 0 @$
(57) 7 @ & 5 7 ! - #15 & 8 5 \ LL]16 resulted in 7 7 8
(58) + + + 6$ + @ 8 + ) showed no + 5 & . Q 5+ &
(59) 4 + + ( ; ! @=L =<. 5+
(60) N
(61) / 0 +
(62) intervention in atherosclerosis related disorders..
(63) Part I: Targeting the Unstable Atherosclerotic Plaque. + 7 & + +
(64)
(65) . & 5
(66) (( ) $ 9 & 8 @
(67)
(68) 7 + $ _ 7 7 @ ! = 8+ & 6$. MATERIAL AND METHODS Animals. 7 7
(69) 7 & $ Male ApoE ) /= + 0 (( ) /< =G 70 7 5
(70) 5
(71) +$ 7 5 + $ O$G@` =@` 5 /# #& # *\0 7 7 & ad libitum throughout the $ Radiolabeling chemokines. 5 /! =Y - # #_ =Y =O f GO fj J *#0 7 5 7 =G@ / N - 0 /! =Y - #0 /
(72) 0 " J ! / #_ =Y
(73) 0$ 125 5 " J 7 5
(74) $ 5 5 & 5 /"#0 & $ Induction of atherosclerotic lesions and temporal gene expression analysis. Male LDLr ) 7 9 + O$G@` =@` 5 /# #& # *\0$ 7 7
(75) 7 + 5+
(76) + & & 5 & +19. O G > ; < =O 7
(77) 7 8 O @ + 7
(78) ' 6. $ - 7 N 7 56 + 7 -$ 9 8 7
(79) 5+ #&B# /( - 0 +$ 7 elative & &
(80) / {O0$. 94.
(81) Targeting Chemokine Receptors during Atherogenesis. In vivo studies: chemokine kinetics, and accumulation by plaque. ! = + ) mice with advanced atherosclerotic lesions were & + Q 7 =| 5 5 /-{D) 0$ ! Q 7 }125 ~ 5 "# & $
(82) 1min, blood samples were taken to determine initial serum concentration and at 56 /@ =O =@ DO
(83) 0 5 7 + 8$ ! 7 5
(84) = 7 5 7 8$ ?
(85) 7 7 +'
(86) & + /7' =>LO - 0$ 7
(87) Q
(88) $ 7
(89) + 8
(90) ? ? +'
(91) + 5+ $ +
(92) 7 6 8 5+ #
(93) 7 /" *#0
(94) 5 5+ Q
(95) 5 }125 ~ 5 "#$. Chapter 4. Statistical Analysis H #$$!$ 7 # 7 & $ &
(96) O$O@ 7 8 $. 95.
(97) Part I: Targeting the Unstable Atherosclerotic Plaque. RESULTS
(98)
(99)
(100)
(101) . atherogenesis.
(102) + 7
(103) 6 &+ 7 &
(104) 6 &
(105) & 5+ + +$ (( ) /-{>=0 7 9 +
(106) 7 5+ 5
(107) + & +$ #5
(108) /-{; L0 7 8
(109)
(110) /O G > ; < =O 70
(111) $ 7 & 6 - 7
(112) + +$. CD68 MMP 1 4. B. MS R 2 A rg inas e -1. 20.0. * 15.0 10.0. * *** **. 5.0 0.0. **. ***. 0. 2. ** *** *** * 4. 6. * ** ** ** 8. *. Relative Expression (A.U.). Relative Expression (A.U.). A. CD3 CD4. * 1.0. 0.0. 10. CD8 F o xp 3. 2.0. 0. 2. 4. Weeks. D. IC A M-1 vW F. C D 3 1 (P E C A M-1 ) C ad he rin. 3.0. Relative Expression (A.U.). Relative Expression (A.U.). C. *. * 2.0. 1.0. 0.0. 0. 2. 4. 6 Weeks. 6. 8. 10. 6. 8. 10. Weeks. 8. 10. A C T A -2 S MT N. S M2 2 a D e s m in. 1.5. 1.0. 0.5. 0.0. 0. 2. 4 Weeks. Figure 4.1
(113)
(114)
(115)
(116)
(117)
(118) .
(119)
(120)
(121)
(122) Atherosclerotic lesions were
(123)
(124) mice on a western-type diet by placement of slightly constrictive collars around both !" # $ %!&' & ()*
(125) $ &' +
(126) ,$ &.' &'/ . 0 formation macrophages were determined. (A) 2 ' 3 4 56
(127) % 27(8 229%:;(B)
(128) ' 34 <4 :4 6=$ <;(C)'34 <% '
(129) % >4*2
(130) %"4' ?. = ?=";(D) vascular smooth 3
(131) '8 *4 *
(132) 8"7288 ' 7288"' 72 )"( $ $ '& !" ' @72 *p<0.05, **p<0.01 and ***p<0.001). 96.
(133) Targeting Chemokine Receptors during Atherogenesis. 97. Chapter 4. _ 7 & +' 5
(134) Q 5 6
(135) / ;< =;D = !#G !!=> -?#0 + + / D > < _ D0 / 0 / D= ! = H &9_0 & & + / G #!GG 0$ ;< !! => 7 + / @
(136) 0
(137) $ &
(138) 7 G 7
(139) !!=> /
(140) 0 7
(141) ;<
(142)
(143) < 7 7 !!=> &+ . & $ ! = /!G 0 & G /!#Gj !G0 7 & G 7
(144) .
(145) !!=> + & 57. 7 > =O /8 >$=0$ !G =;D != /-?#0 7 . & / 7 0$ +
(146)
(147) D > _ D + 6 . $ ? + < 7 5 8 7 7
(148) $ H
(149) 5 H 95
(150) /&9_0 ) = / ! =0 H 7 . initiation and progression in LDLr ) $ + & = / ! =0 7
(151)
(152)
(153) 8 G < 7
(154) /8 >$= 0$ H #! /#! -0 Y G / G0 GG /#!GGY0 7 5+ 7 7 7
(155) . 5 & /8 >$=0$ 7 '
(156)
(157) / 5=0 56 + in vivo
(158) 7 5 $ + 5 5&
(159)
(160) $ (D /! =0 7 7 7
(161) > 7
(162)
(163)
(164) $ 7 ; (D . 7 =O /_ >$G0$ 5 7 5&
(165) B (=G 7 G 7 7 > 56 7 ; =O /_ >$G0$ 7 (D B (=G (@ 7 &+ & 6
(166) /_ >$G"0$ . & (== 7
(167) /_ >$G 0$ ?
(168) & + @ B > 7 8 5 5
(169) /_ >$D .
(170) Part I: Targeting the Unstable Atherosclerotic Plaque. 9.0. Relative Expression CCL5 (A.U.). Relative Expression CCL3 (A.U.). "0$
(171) 5 G 7
(172) 7 5+ > 7 56
(173) 7 ; 7$
(174) = G D > & +
(175)
(176) 8 =O 7
(177) 6 & /_ >$D 0$. A. 8.0 7.0 6.0 5.0 4.0 3.0. ***. **. 2.0 1.0 0.0. 0. 2. 4. 6. 8. 10. B. 2.5 2.0. *. 1.0 0.5 0.0. 0. 2. 4. Relative Expression CXCL12 (A.U.). Relative Expression CCL11 (A.U.). C 1.5. 1.0. 0.5. 0. 2. 4. 6. 8. 10. 8. 10. weeks. weeks. 0.0. *. *. 1.5. 6. 8. 10. D. 7.0. ***. 6.0 5.0 4.0 3.0 2.0 1.0 0.0. 0. 2. 4. weeks. 6. weeks. A. *. ***. 4.0. * 3.0. ***. *. 2.0 1.0 0.0. 0. 2. 4. 6. 8. 10. Relative expression (A.U.). C 1.5. 1.0. 0.5. 0. 2. 4. 6. weeks. 98. 2.5. B **. 2.0. *. 1.5 1.0 0.5 0.0. 0. 2. 4. 6. 8. 10. weeks. weeks. 0.0. Relative Expression CXCR4 (A.U.). Relative Expression CCR5 (A.U.). Figure 4.2
(178)
(179)
(180)
(181) .
(182) .
(183) 2
(184) & '$ +& $ &'& '' # :< $ .'
(185) '& 44 <(A)44 E(B)44 %%(C) and 4F4 %8(D)($ $ & !" '@72 (*p<0.05, **p<0.01 and ***p<0.001). 8. 10. Figure 4.3
(186)
(187)
(188) .
(189)
(190) G ' $ .# :%'
(191) & $ &' , $ & $ &44(E(A) and CXCR4 (B),$ ''' # && $ 44(% 44(8 CCR3 and CCR4)(C) ( $ $ & !" '@72 (*p<0.05, **p<0.01 and ***p<0.001).
(192) Targeting Chemokine Receptors during Atherogenesis. MIP-1alpha and CCL5 accumulate in advanced atherosclerotic lesions.. B. 100. Serum Clearance (% of I.D.). Serum Clearance (% of I.D.). A. 75. 50. 25. 0 0. 10. 20. 30 40 Time (min). 50. 60. 100. 75. 50. 25. 0 0. 10. 20. 30 40 Time (min). 50. 60. Figure 4.4 Serum clearance data for radiolabeled chemokines in aged ApoE-/mice. 7 +'
(193) &
(194) 5+ + 7 6 7 & /125 0 & + Q = + ) mice. During 1h incubation, serum 7
(195)
(196) /= @ =O GO DO 0
(197) 8 $ # 7 +'
(198) $ (A) Serum clearance
(199) ( D /"44 E ). (B)7 & 44 %% {"4F4 %8 z" '@72. 99. Chapter 4. J& 8 @ B > 7 & 6 7 56 + &
(200)
(201) &+
(202) & ) $
(203) /$$ (D (@ B (=G0 7
(204) + $ D (== 7 7 6 & $ 7 5 7 }125 ~ 5+ / (@ (D0 " . J / (== B (=G0 =| 5 7 & + Q = + ) mice with advanced atherosclerotic plaques. Serum + 5
(205) 5 7 /8 >$>0$ (D (@ 7 +
(206) 5
(207) &
(208) &+ @ =G $ (== B (=G + 5 />O @O`
(209) Q 0 &+ 7
(210) 7 & 7 8 + 57 =O ;O .
(211) Q 7 7$ (D (@ &+ =
(212) Q 7 7 & 5 /$$ = > @0$ (& + 7
(213) 5 /_ >$@0$.
(214) Chemokine accumulation (% of I.D./g). Part I: Targeting the Unstable Atherosclerotic Plaque. 4.0 10 3.0. 2.0 5 1.0. 0. CCL3. CCL5. CCL11. CXCL12. 0.0. CCL3. CCL5. CCL11. CXCL12. Figure 4.5
(215)
(216)
(217)
(218)
(219)
(220)
(221) -/mice. 7 +'
(222) &
(223) 5+ + 7 6 7 & /125 0 & + Q = + ) $
(224) = 5
(225) 7 & +'
(226)
(227) $ /0 ? 7 $$ (& /7 50 # /+ 50 \ + / + 50 ( /5 50$ /"0 ? + 7 $$ /7 50 5 7 /+ 50 / + 50 + /5 50$ 7 #!$. _ + 7 & & &
(228) ) $ - 8
(229)
(230) 7 .
(231) 6
(232) Q
(233) ? ? 57
(234)
(235) /_ >$;" 0$
(236) 5 5+ + & ! =Y /GD<$; =G$;` 6 j O$O@0 (@ /G<>$@ =O=$G`j {O$==0 6
(237) )
(238) 5 "# /=OO$O D@$>`0 /8 >$; 7 0$ 7 6
(239) D (== 8 + 6 7 B (=G 7 5. 8 + 5 5+ 6$. Plaque accumulation (%). 500. 400. 300. *. 200. 100. 0. BSA. CCL3. CCL5 CCL11 CXCL12. Figure 4.6 Accumulation of different chemokines in advanced aortic atherosclerotic lesions of in aged ApoE-/- mice. 7 +'
(240) &
(241) 5+ + 7 6 7 & /125 0 & + Q = + ApoE ) /-{D) 0$
(242) = 5 7
(243) +
(244) ? ? /j 0$ #56 + 7
(245) 5+ + & &' 7 + ]>OO /j 7 0$ * + 7 6 8
(246) 5+ ? ? 7
(247) 5 "# 7 & /"0$
(248) }125 ~ "# /=OO`0 7 #!$ /O$O@0. 100.
(249) Targeting Chemokine Receptors during Atherogenesis. DISCUSSION. 101. Chapter 4. & 7 . cardiovascular research and our knowledge on their role in the pathogenesis
(250) + $ 8
(251) & 5
(252) & 6 5+ $ +
(253) /
(254) 0
(255)
(256) + / 0 7 5 & 7 7. 6 $ + 7 & 8 during atherogenesis in diet induced carotid atherosclerosis in LDLr ) =]. Established
(257) Q 6 / ;< =;D = !#G !!=> -?#0 + + / D > < _ D0 / 0 / D= ! = &9_0 & / G #!GG 0 7 & 6
(258)
(259) $ 8 7 +
(260) &+ +
(261) 7 7
(262) 7 + 7 8 + .
(263) = !G + 4&
(264)
(265) +
(266) & + 20. Moreover there was a dramatic !! => 7 8 5
(267) + &&
(268) macrophages21 5+ 8 . ! =$
(269) &
(270) + . 5
(271) & 5 5 5+ 4
(272) macrophages. 6
(273) 5
(274) &
(275) 5 @ B > 7
(276) = > 7 8 + /_ >$G0$ " 7 7 7 7
(277) $ &
(278)
(279) @
(280) $ _ 5
(281) @ 7 - # @ 5 / \ LL]0 8 + lesion progression in atherosclerotic mice15, 16 7 @ 8 + 7 . &
(282) 11. Quinones et al.18, reported that ApoE ) CCR5 )
(283) & @ + 5 + &
(284) $
(285) B > 5 $ " $22 showed that
(286) 5 +
(287) /! _0 7 B > & +
(288)
(289) $ &+ B > 5.
(290) Part I: Targeting the Unstable Atherosclerotic Plaque. 7 &
(291)
(292) GD + 5+ . 6 5+ #! mobilizationG>$ ! & &
(293) #_= & 7 in patients with unstable angina pectoris25$
(294) + 5 @ B > 7 &+ 6 .
(295) + @
(296) 5 & B > +
(297) $ - +
(298) @ B > 5
(299) & (D B (=G 7 G 7
(300) + > 7 5' / B (=G0
(301) / (D0 57 ; =O 7$ (D ! 4 + = /! =Y0 5+ &+
(302) + + and platelets6, 8 &
(303) = @$ (D 5 5&
(304) 26$ B (=G # & _ = /#_ =Y0
(305) B >$ 7 B (=G 7 + 7 & 7 5 &+ 5+ & . atheromasGL$ @ (@ - # /Regulated on Activation, -ormal T cell E S0 7 + 7 + 5 7 & 8 /=D0$ 5+ + +
(306) = @ 7 5
(307) + + + 28, 29 $ _ +
(308) (== 7
(309)
(310) $ (== / 0 + 5+ #! + & D & 7 7 + 6 5' DO$ ( (D 7 7 5 present in human atherosclerotic lesions=O D=.
(311) & 5 @ B > 7 & 6 7
(312) $ ? 5 8
(313) (D (@ 7 + 7 5 5+
(314) 5 / = @0$ (== 7
(315) 8 +
(316) D +
(317)
(318) 7 &+ 7 D & cellsDG.
(319) 6 5+ /=>0 (D / (@0 5 @
(320) &
(321) $&$ injection.. 102.
(322) Targeting Chemokine Receptors during Atherogenesis. & &
(323) @ B > 5
(324) = > B D 6
(325) +
(326)
(327) & &+
(328) & 4 + 6$
(329) 7 Q @ - # ! = + . advanced atherosclerotic lesions in aged ApoE )
(330) & Q 7
(331) B > B D 7 + +
(332) 5+ 6$ N& 5
(333) @ 6 7 @ + + & 5 6 5
(334) 8+
(335) 6 5' $. Chapter 4 103.
(336) Part I: Targeting the Unstable Atherosclerotic Plaque. REFERENCES =$. 104. ( %$ $ Nature. GOOOj>OLGDD G>=$. G$. (55+ $ 4 $ Nature. GOOGj>GO<;< <L>$. D$. $ 4 + $ N Engl J Med. =]]]jD>O==@ =G;$. >$. ! " 7 9 $
(337) &
(338) & 8 $ Circulation. GOOOj=O=GG]O GG]@$. @$. - 9 # " 7 %( $ *
(339) H ! = ! = 8 $ Arterioscler Thromb Vasc Biol. =]]<j=<<>G <@=$. ;$. 95 $ $ Circ Res. GOO@j];;=G 616.. L$. J (+ \_$
(340) &
(341) $ Trends Cardiovasc Med. GOO>j=>=< GG$. <$. \Q& ? % # & " % " % %9$ Chemokines and 6 7 Curr Pharm Des. GOOLj=D=OD] =O@G$. ]$. " H ! _ #
(342)
(343) #$ 8
(344) $ Thromb Haemost. GOOLj]LL=> LG=$. =O$. 95 # 5 $ +
(345) . in atherosclerotic vascular disease. Arterioscler Thromb Vasc Biol. GOO>jG>=]]L GOO<$. ==$. " H ( #
(346)
(347) # # "' & \ " _ N ( 7 " ! _ 95 $ @ 5 = 8 + &
(348) $ Arterioscler Thromb Vasc Biol. GOOLjGLDLD DL]$. =G$. & J " J # J ! & . & % ( % & # #+ 9 \ $ # . 6 5+ $ J Clin Invest. GOOLj==L;=; 626.. =D$. ( _5 " # \ & & J
(349) J # Q \" "Q " ( %N !+ . "+ H % ! " \ + H _ !%$ N 8 & +
(350) 5 + & & + 5 +$ Circulation. GOO@j===D>>D D>@G$. =>$. _ 9$ G / G0
(351)
(352) ' $ Microcirculation. GOODj=OG@] G;>$. =@$. H - \7 " N ! _ % 9
(353) ! _$
(354) - # 6
(355) $ Circ Res. GOO>j]>G@D 261.. =;$. & 9 Q % J J J H _Q7 J & " % \ %$ J H + 5 \ LL] 7 + 8 $ Arterioscler Thromb Vasc Biol. GOO@jG@G;>G G;>L$. =L$. \' 9 7 . ! N& N Q ## ( ! -$ @ 8 + & +
(356) $ Atherosclerosis. GOODj=;LG@ DG$. =<$. . ! ! ' JN % ' _ + ! 9 ? \ J ( _ N \' 9 Q #\ Q ##$ @ 4 stage atherosclerosis. Atherosclerosis. GOOLj=]@]G =OD$. =]$. & %J & HQ "% J 5 \ !! J& (! & " % ". $
(357) 6 )
(358) &
(359)
(360) @D$ Circulation. GOOGj=O@GO;> GOLO$. GO$. \ 7 ! "+ # N H !$ '
(361) (( receptor knockout mice: macrophage accumulation correlates with rapid and sustained
(362) ! =)%$ Atherosclerosis. GOOOj=>]DGD DDO$. G=$. % %( # -75+ N" ! -75+ $ ( 7 5
(363) D => & 8 5
(364) + &&
(365) $ Arterioscler Thromb Vasc Biol. GOO<jG<=;>L =;@D$.
(366) Targeting Chemokine Receptors during Atherogenesis " % \ ( J N + %( \ 7 ! N & # 5 ( ( \ _ 7 N N'' \. ! # " J+ !% 95 $ ! _ .
(367) B 4 + and atherogenic cell recruitment. Nat Med. GOOLj=D@<L @];$. GD$. " Q 5 # "' & \ ! # \ # 5 # ! # ? " . % _ " 95 $ &
(368) B >) B =G &
(369) $ Circ Res. GOO<j=OGGO] G=L$. G>$. + %! ( 9 % + ! # " $ 57 B >) &
(370) = H( >)H ! = 7+ 5 marrow. J Immunol. GOO]j=<G;O> ;=G$. G@$. %\ 9 * ! _ JN J ! J& " _ ## N ( $ # &
(371) = 5 4 + 5'
(372)
(373) $ Circulation. GOOGj=O;D; >G$. G;$. 9 %- - - # N # %$ (
(374) 4 + + 6$ J Atheroscler Thromb. =]]>j= # =#=O =D$. GL$. 5 # #+ ! _ # & N\ (55+ ( $ &
(375) = + 6$ Circ Res. GOOOj<;=D= =D<$. G<$. \ ! ". "
(376)
(377) # # % $ - # 4 + = &
(378) +$ J Exp Med. =]]Gj=L;=><] =>]@$. G]$. " & J ! #_ " 9 95 $
(379)
(380) &
(381)
(382)
(383) & + 4 4 7 $ J Leukoc Biol. GOO@jL<>D@ >>=$. DO$. " % # ( \ & " % 95 " $ Perivascular mast cells promote atherogenesis and induce plaque destabilization in apolipoprotein 8 $ Circulation. GOOLj==@G@=; G@G@$. D=$. J+ \% (+ ! %J _ \. + # N %_ # & NJ (55+ ( $ ?&
(384) D +
(385) + & 7+
(386) & 4 $ Circulation. GOOOj=OGG=<@ G=<]$. DG$. # JJ B _ N# 9 #" B 9J$ D 5 + 5 7+ 4 &
(387) $ Acta Pharmacol Sin. GOO;jGL=@]> =@]]$. 105. Chapter 4. GG$.
(388)
(389)
GERELATEERDE DOCUMENTEN
Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden.. Note: To cite this publication please use the final
Detection of amyloid plaques in mouse models of Alzheimer’s disease by magnetic resonance imaging.. Apostolova
More precisely, an upper bound for the variance of the test statistic R N ∗ is realized by the one-dimensional Moore-Rayleigh null hypothesis, whose distribution is similar to the
The results on simulated data show that the MR3 outperforms permutation tests on Hotelling’s T 2 test (pHT2) in the detection of difference of mean in small sample sizes, since the
Since expression of Serpins may facilitate the immune escape of HLA positive tumors, we next analysed the effect of Serpin expression on survival in cases with normal/partial
Peripheral blood cells were stained with HLA-A2.1 tetramers containing the tyrosinase368–376 peptide followed by staining with a panel of lineage antibodies, as described in
Blades and blade fragments seem to have been especially used for longitudinal motions, mainly on plant material (7/12). Flake and flake fragments are used in different motions on
This shape also occurs in the combination artefacts (see below). The shape is the result of intensive use in a repetitive abrasive motion, carried out from different angles. In
